JP6966176B2 - Antibodies specific for human poliovirus receptor (PVR) - Google Patents

Description

The present invention relates to the field of immunotherapy, which relates to antibodies and fragments thereof that bind to protein poliovirus receptors, polynucleotide sequences encoding these antibodies, and hybridoma cells that produce these antibodies. The present invention further relates to therapeutic and diagnostic compositions comprising these antibodies and methods of treating and diagnosing diseases, especially cancers, using these monoclonal antibodies.

Tumor immunotherapy involves, for example, an antitumor immune response by treatment with antigen-specific antibodies on tumor cells, fusion of antigen-presenting cells with tumor cells, or specific activation of antitumor T cells. Used to generate and grow. The ability of a patient to replenish immune cells (eg, T cells) with tumor cells provides a therapeutic means of combating cancer types and metastases previously considered incurable.

A T cell-mediated immune response involves multiple contiguous steps regulated by the equilibrium between co-stimulatory and co-suppressive signals that control the magnitude of the immune response. Suppressive signals, called immune checkpoints, are also essential for maintaining self-tolerance and limiting immune-mediated concomitant tissue damage. When the infection or immune induction is removed, exacerbated, or persisted, these signals change, and those changes affect the T cell response and reshape the immune response.

Immune checkpoint protein expression is regulated by the tumor. For example, upregulation of a programmed death-1 ligand (PD-L1) on the surface of cancer cells causes T cells through binding to PD-1, which may otherwise attack these tumor cells. By inhibiting, they allow them to escape the host immune system. Therefore, immune checkpoints represent a significant impairment in the activation of functional cell-mediated immunity against cancer. Therefore, antagonist antibodies specific for suppressor ligands on immune cells are considered viable anti-cancer agents and are being evaluated in the clinic (eg, nivolumab and pembrolizumab). Another example of an immune checkpoint molecule is a T cell immune receptor with Ig and ITIM domains (TIGIT). TIGIT is a co-suppressive molecule expressed on a variety of immune cells, including T cells and natural killer cells (NK cells). TIGIT binds to the poliovirus receptor (PVR) with high affinity.

The poliovirus receptor (PVR), also called CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. It was previously described as a tumor antigen and its expression is similar to pancreatic cancer (Nishiwada et al., Anticancer Research 2015, 35 (4): 2287-97), polymorphonuary glioblastoma, medulloblastoma, and It is also up-regulated in neuroepidermal cancers, including colorectal cancer (Solecki et al., J. Biol. Chem. 2002, 277: 25679-700) and is also a promising target for therapeutic intervention. Has been described. PVR is a serum-induced activation of Ras-Raf-MEK-ERK signaling, up-regulation of cyclins D2 and E, and down-regulated p27Kip1, and ultimately the period of G0 / G1 phase of the cell cycle. It is known to enhance the shortening of PVRs (Kakunaga 2004, J. Biological Chemistry, 279, 36419-36425), for which blocking PVR on tumor cells is expected to reduce tumor cell viability. PVR also plays a key role in angiogenesis, regulating VEGF-induced angiogenesis and VEGFR2-mediated Rap1-Akt signaling pathways by controlling VEGFR2 interactions with integrin α (v) β. It has been suggested that (Kinugasa et al., 2012, Circ Res. 2012, 110 (5), 716-26). In addition, PVR was complexed with IGF1R and involved in Met signaling, and inhibition of complex formation reduced cell viability and angiogenesis (Lee et al., Scientific Reports 2014, 20, 4). , 7139).

The involvement of PVR in metastasis was demonstrated by injecting cancer cells into the tail of mice and measuring metastasis to the lung. Up-regulated PVRs in cancer cells transinteract with their antireceptors in platelets, and this trans-interaction enhances the metastasis of cancer cells to the lung (Morimoto et al., Oncogene (Morimoto et al., Oncogene). 2008) 27, 264-273).

U.S. Patent Application No. 20070041985 discloses a molecule that specifically binds to at least one intracellular or extracellular domain of PVR or any derivative thereof, wherein the molecule refers to PVR or any derivative thereof. It has the ability to regulate receptor-mediated attachment, transport, and / or invasion behavior of expressed cells.

US Pat. Antibody fragment) is provided. The molecule can be used to treat cells with metastatic potential, metastasis, and cancer.

PCT Application Publication No. 2006/1246667 discloses the regulation of protein zB7R1 (TIGIT) by a monoclonal antibody that blocks TIGIT that binds to its ligand PVR.

Further, more effective, and specific, it enhances cells of the immune system that attack cells infected with tumors or viruses by inhibiting PVR that binds to TIGIT, alone or in combination with other agents. There is an unmet need to provide safe and / or stable drugs.

The present invention recognizes the poliovirus receptor (PVR), prevents its binding to T cell immunoreceptors with Ig and ITIM domains (TIGIT), and on lymphocytes such as natural killer (NK) cells and T cells. Provided are antibodies and fragments thereof that inhibit the inhibitory activity of. Antibodies of anti-PVR disclosed herein can bind to PVR present in cancer cells. These antibodies and fragments thereof are characterized by having a unique set of complementarity determining region (CDR) sequences, high affinity, and high specificity for PVR, as stand-alone therapies, and other. In combination with anti-cancer agents, it is useful in cancer immunotherapy to combat tumor immunoavoidance. Antibodies are also useful in treating viral infections.

It is disclosed herein that the high affinity anti-PVR antibody disclosed herein blocks the TIGIT-PVR interaction and restores the activity of T and NK cells. The antibody showed high specificity for human PVR. These properties make the monoclonal antibodies (mAbs) of the invention a valuable candidate for use in cancer immunotherapy, allowing low dose administration with few side effects.

Advantageously, the anti-PVR mAbs according to the present invention can induce T cell proliferation superior to anti-PD-1 and CTLA-4 mAbs in PD-L1 of the in vitro model (A549). Inducing effects were shown for peripheral mononuclear blood cells (PMBC) and purified CD4 and CD8 T cells. In addition, PVR mAbs were able to induce NK cell activation in most of the target cells tested. In addition, some of the anti-PVR antibodies described herein have comparable anti-cancer activity in vitro to those of known agents (Erbitux® currently used in treatment). .. In addition, some of the anti-PVR antibodies described herein show synergistic effects when combined with additional anti-cancer agents such as anti-PD-1 and CTLA-1 antibodies and the dermal growth factor receptor (EGFR). rice field. In addition, some of the anti-PVR antibodies have been found to induce antibody-dependent cellular cytotoxicity (ADCC). It is also disclosed that some of the anti-PVR antibodies described herein do not have a blocking effect on DNAM1 co-stimulation signaling and therefore have no detrimental effect on other immune-inducing signals. There is.

Interestingly, despite the high sequence similarity between human and rodent PVR sequences, the antibodies of the invention are highly specific for human PVR.

Unexpectedly, it is also disclosed that some chimeric monoclonal antibodies containing human constant strands showed enhanced effects on immune cell activation compared to comparable mouse monoclonal antibodies.

Some of the anti-PVR mAbs described herein were able to reduce tumor cell viability in an immune-independent manner by blocking PVR on tumor cells. Not expected to be bound by any mechanism of action, this activity has been suggested to be due to PVR's ability to shorten the G0 / G1 phase of the cell cycle.

According to one embodiment, the invention provides an isolated monoclonal antibody that binds to a poliovirus receptor (PVR), or an antibody fragment thereof that comprises at least an antigen binding moiety, and the isolated antibody or antibody. Fragments are selected from the following group:
i. Three CDRs of the heavy chain (HC) variable region containing SEQ ID NO: 77, three CDRs of the light chain (LC) variable region containing SEQ ID NO: 79, or at least 90% of the antibody or fragment sequence described above. Its analog or derivative with sequence identity;
ii. Three complementarity determining regions (CDRs) of heavy chain (HC) variable regions containing SEQ ID NO: 69, and three CDRs of light chain (LC) variable regions containing SEQ ID NO: 71, or the above antibodies. Or an analog or derivative thereof having at least 90% sequence identity with the fragment sequence; and
iii. Three CDRs of the heavy chain (HC) variable region containing SEQ ID NO: 73, three CDRs of the light chain (LC) variable region containing SEQ ID NO: 75, or at least 90% of the antibody or fragment sequence described above. Its analog or derivative having sequence identity.

Antibodies comprising CDR sequences contained in heavy and light chains homologous to SEQ ID Nos: 2, 10, 18, 26, 34, or 42 are included within the scope of the invention. According to some embodiments, SEQ ID NO: 2 is interchangeable with SEQ ID NO: 69. According to some embodiments, SEQ ID NO: 10 is interchangeable with SEQ ID NO: 71. According to some embodiments, SEQ ID NO: 18 is interchangeable with SEQ ID NO: 73. According to some embodiments, SEQ ID NO: 26 is interchangeable with SEQ ID NO: 75. According to some embodiments, SEQ ID NO: 34 is interchangeable with SEQ ID NO: 77. According to some embodiments, SEQ ID NO: 42 is interchangeable with SEQ ID NO: 79.

There are several methods known in the art for determining the CDR sequence of a given antibody molecule, but there is no standard clear method. Determining the CDR sequence from the variable regions of the heavy and light chains of an antibody can be done according to any method, including but not limited to the method known as KABAT, Chothia, and IMGT. A selected set of CDRs may contain sequences identified by more than one method, i.e., some CDR sequences may be determined using KABAT, eg, determined using IMGT. May be done.

According to some embodiments, the isolated monoclonal antibody or fragment is described by the CDR sequence of the monoclonal antibody labeled anti-PVR 4E5 (or hPVR.07), ie, SEQ ID NO: 69. It contains three CDR sequences contained in the heavy chain variable regions and three CDR sequences contained in the light chain variable regions described by SEQ ID NO: 71.

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 containing the sequence GFDFSRYW (SEQ ID NO: 4). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR2 containing the sequence EIHPDSSKINYTPSQ (SEQ ID NO: 6). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR3 containing the sequence PDGNYNALDYW (SEQ ID NO: 8).

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 containing the sequence RYW. According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 containing the sequence RYWMT (SEQ ID NO: 80). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR3 containing the sequence PDGNYNALDY (SEQ ID NO: 82).

According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) HC CDR1 containing sequence GFDFSRYW (SEQ ID NO: 4); (ii) sequence: EIHPDSSKINYTPSQ (SEQ ID). HC CDR2 containing NO: 6) and HC CDR3 containing (iii) sequence: PDGNYNALDYW (SEQ ID NO: 8).

According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) HC CDR1 containing sequence RYW; (ii) sequence: HC CDR2 containing EIHPDSSKINYTPSQ (SEQ ID NO: 6): And (iii) sequence: HC CDR3 comprising PDGNYNALDY (SEQ ID NO: 82).

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 comprising the sequence KASQDVGTAVT (SEQ ID NO: 12). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 comprising the sequence WASTRHT (SEQ ID NO: 14). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 comprising the sequence QQYSRYPYT (SEQ ID NO: 16).

According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) LC CDR1 comprises sequence KASQDVGTAVT (SEQ ID NO: 12) and (ii) LC CDR2 comprises sequence: WASTRHT (SEQ ID NO: 14) is included, and (iii) HC CDR3 comprises sequence: QQYSRYPYT (SEQ ID NO: 16).

According to some specific embodiments, the isolated monoclonal antibody or fragment comprises a heavy chain CDR1 sequence comprising sequence: GFDFSRYW (SEQ ID NO: 4), sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6). Heavy chain CDR2, sequence: heavy chain CDR3 containing PDGNYNALDYW (SEQ ID NO: 8), sequence: light chain CDR1 containing KASQDVGTAVT (SEQ ID NO: 12), sequence: light including WASTRHT (SEQ ID NO: 14) Chain CDR2) and sequence: Light chain CDR3 containing QQYSRYPYT (SEQ ID NO: 16), or its analog containing at most 5% amino acid substitutions, deletions, and / or insertions in the hypervariable region (HVR) sequence. I'm out.

According to some specific embodiments, the isolated monoclonal antibody or fragment comprises one set of six CDR sequences consisting of: i. Heavy chain CDR1, ii. With sequences selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 80. Heavy chain CDR2, iii. With a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 81. Heavy chain CDR3, iv. Light chain CDR1, v. Light chain CDR2, vi. Light chain CDR3 having the sequence described by SEQ ID NO: 16.

The isolated monoclonal antibodies or fragments of some particular embodiments are SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, and. Includes one set of six CDR sequences consisting of SEQ ID NO: 16.

The isolated monoclonal antibodies or fragments of other specific embodiments are SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ. Includes one set of six CDR sequences consisting of ID NO: 16.

According to some embodiments, the isolated monoclonal antibody or fragment has at least 90% sequence identity with the heavy chain variable region described by SEQ ID NO: 69, or the heavy chain variable region sequence thereof. Includes its analogs or derivatives.

According to some embodiments, the analog of SEQ ID NO: 2 is a heavy chain variable region having the sequence described by SEQ ID NO: 69.

According to some embodiments, the isolated monoclonal antibody or fragment has at least 90% sequence identity with the light chain variable region described by SEQ ID NO: 71, or the light chain variable region sequence thereof. Including analog.

According to some embodiments, the analog of SEQ ID NO: 10 is a light chain variable region having the sequence described by SEQ ID NO: 71.

According to certain embodiments, the isolated monoclonal antibody or fragment is a heavy chain variable region having the sequence described by SEQ ID NO: 2 or SEQ ID NO: 69, as well as SEQ ID NO: 10 or SEQ ID. It comprises a light chain variable region having the sequence described in NO: 71, or an analog thereof having at least 90% sequence identity with the light chain and / or heavy chain sequence.

The present invention further includes antibodies or antibody fragments capable of binding with high affinity to epitopes within the human PVR protein to which the monoclonal antibody (mAb) 4E5 binds.

According to other embodiments, the isolated monoclonal antibody is contained in the CDR sequence of the monoclonal antibody labeled 7D4 (or hPVR.01), i.e. the heavy chain variable regions described by SEQ ID NO: 73. Contains three CDR sequences and the three CDR sequences contained in the light chain variable regions described by SEQ ID NO: 75.

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 containing the sequence GYTFTEYTMH (SEQ ID NO: 20). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR2 containing the sequence GIDPNNGGTNYNQNFKG (SEQ ID NO: 22). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR3 containing the sequence VIPLEY (SEQ ID NO: 24). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) HC CDR1 comprises sequence GYTFTEYTMH (SEQ ID NO: 20) and (ii) HC CDR2 comprises sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22) is included, and (iii) HC CDR3 comprises sequence: VIPLEY (SEQ ID NO: 24).

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 containing the sequence EYTMH (SEQ ID NO: 83).

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 comprising the sequence KASQNVYTNVA (SEQ ID NO: 28). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 comprising the sequence SASYRYR (SEQ ID NO: 30). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 comprising the sequence QQYNSYSPLA (SEQ ID NO: 32). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) LC CDR1 comprises sequence KASQNVYTNVA (SEQ ID NO: 28) and (ii) LC CDR2 comprises sequence: It comprises SASYRYR (SEQ ID NO: 30), and (iii) HC CDR3 comprises sequence: QQYNSYPLA (SEQ ID NO: 32).

According to some specific embodiments, the isolated monoclonal antibody is a heavy chain CDR1 containing the sequence GYTFTEYTMH (SEQ ID NO: 20), a heavy chain CDR2 containing the sequence GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), Sequence: Heavy chain CDR3 containing VIPLEY (SEQ ID NO: 24), Sequence: Light chain CDR1 containing KASQNVYTNVA (SEQ ID NO: 28), Sequence: Light chain CDR2 containing SASYRYR (SEQ ID NO: 30), And Sequence: Light chain CDR3 containing QQYNSYSPLA (SEQ ID NO: 32), or an analog thereof containing at most 5% amino acid substitutions, deletions, and / or insertions in the HVR sequence.

According to some specific embodiments, the isolated monoclonal antibody or fragment comprises one set of six CDR sequences consisting of: Select from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 83. CDR1, SEQ ID NO: 24 having the sequence described in, heavy chain CDR3 having the sequence described in SEQ ID NO: 24, light chain CDR1, SEQ ID NO: 30 having the sequence described in SEQ ID NO: 28. Light chain CDR2 having, and light chain CDR3 having the sequence described by SEQ ID NO: 32.

According to some specific embodiments, the isolated monoclonal antibody or fragment is SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30. , And a set of six CDR sequences consisting of SEQ ID NO: 32.

The isolated monoclonal antibodies or fragments of other specific embodiments are SEQ ID NO: 83, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ. Includes one set of six CDR sequences consisting of ID NO: 32.

According to some embodiments, the isolated monoclonal antibody or fragment has at least 90% sequence identity with the heavy chain variable region described by SEQ ID NO: 73, or the heavy chain variable region sequence thereof. Includes analogs or derivatives.

According to some embodiments, the analog of SEQ ID NO: 18 is a heavy chain variable region having the sequence described in SEQ ID NO: 73.

According to some embodiments, the isolated monoclonal antibody or fragment has at least 90% sequence identity with the light chain variable region described by SEQ ID NO: 75, or the light chain variable region sequence thereof. Including analog.

According to some embodiments, the analog of SEQ ID NO: 10 is a light chain variable region having the sequence described by SEQ ID NO: 75.

According to certain embodiments, the isolated monoclonal antibody or fragment is a heavy chain variable region having the sequence described by SEQ ID NO: 18 or SEQ ID NO: 73, as well as SEQ ID NO: 26 or SEQ ID. It comprises a light chain variable region having the sequence described by NO: 75, or an analog thereof having at least 90% sequence identity with the light chain and / or heavy chain sequence.

The present invention further includes an antibody or antibody fragment capable of binding with high affinity to an epitope in a human PVR protein to which mAb7D4 binds.

According to other embodiments, the isolated monoclonal antibody is contained in the CDR sequence of the monoclonal antibody labeled 5B9 (or hPVR.09), i.e., the heavy chain variable region described by SEQ ID NO: 77. Contains three CDR sequences and the three CDR sequences contained in the light chain variable regions described by SEQ ID NO: 79.

According to some embodiments, the isolated monoclonal antibody comprises the complementarity determining region (CDR) sequence contained in the heavy chain variable region described by SEQ ID NO: 34, and SEQ ID NO: 42, SEQ. Included in the light chain variable region sequence selected from the group consisting of ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54. Contains three CDR sequences. Each possibility represents another embodiment of the invention.

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 containing the sequence GYTFSNYWIE (SEQ ID NO: 36). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR2 containing the sequence EIFFPGSGRINGINFNEKFKG (SEQ ID NO: 38). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR3 containing the sequence TKIYGNSFDY (SEQ ID NO: 40). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 containing the sequence SNYWIE (SEQ ID NO: 84).

According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) HC CDR1 comprises sequence SNYWIE (SEQ ID NO: 84) and (ii) HC CDR2 comprises sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38) is included, and (iii) HC CDR3 comprises sequence: TKIYGNSFDY (SEQ ID NO: 40).

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 comprising the sequence KASQDVGTAVV (SEQ ID NO: 44). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 comprising the sequence WASSRHN (SEQ ID NO: 46). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 comprising the sequence QQYSRYPLT (SEQ ID NO: 48).

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 comprising the sequence KASQDVGTAV (SEQ ID NO: 85).

According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) LC CDR1 comprises sequence KASQDVGTAV (SEQ ID NO: 85) and (ii) LC CDR2 comprises sequence: WASSRHN (SEQ ID NO: 46) is included, and (iii) HC CDR3 comprises sequence: QQYSRYPLT (SEQ ID NO: 48).

In a further embodiment, LC CDR2 comprises the sequence described in SEQ ID Nos: 46, 56, 57, 58, 59, or 61. Each possibility represents another embodiment of the invention.

According to some specific embodiments, the isolated monoclonal antibody or fragment comprises a heavy chain CDR1 sequence comprising sequence: GYTFSNYWIE (SEQ ID NO: 36), sequence: EIFFPGSGRINGFNEKFKG (SEQ ID NO: 38). Heavy chain CDR2, sequence: heavy chain CDR3 containing TKIYGNSFDY (SEQ ID NO: 40), sequence: light chain CDR1 containing KASQDVGTAVV (SEQ ID NO: 44), sequence: light chain containing WASSRHN (SEQ ID NO: 46). CDR2, and sequence: light chain CDR3 containing QQYSRYPLT (SEQ ID NO: 48), or an analog thereof containing at most 5% amino acid substitutions, deletions, and / or insertions in the HVR sequence.

According to some specific embodiments, there is an isolated monoclonal antibody or fragment consisting of: heavy chain CDR1, having a sequence selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 84: Selected from the group consisting of heavy chain CDR2 having the sequence described by SEQ ID NO: 38, heavy chain CDR3 having the sequence described by SEQ ID NO: 40, SEQ ID NO: 44 and SEQ ID NO: 85. Light chain CDR1 having a sequence, light chain CDR2 having a sequence described by SEQ ID NO: 46, and light chain CDR3 having a sequence described by SEQ ID NO: 48.

According to some specific embodiments, there are isolated monoclonal antibodies or fragments consisting of: heavy chain CDR1, SEQ ID NO with the sequence described by: SEQ ID NO: 36 or SEQ ID NO: 84. Heavy chain CDR2 having the sequence described by: 38, heavy chain CDR3 having the sequence described by SEQ ID NO: 40, or light chain CDR1 having the sequence described by SEQ ID NO: 44 or SEQ ID NO: 85. , SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61. A light chain CDR2 having a sequence and a light chain CDR3 having a sequence described by SEQ ID NO: 48. Each possibility represents another embodiment of the invention.

According to some embodiments, the isolated monoclonal antibody or fragment has at least 90% sequence identity with the heavy chain variable region described by SEQ ID NO: 77, or the heavy chain variable region sequence thereof. Includes analogs or derivatives.

According to some embodiments, the analog of SEQ ID NO: 34 is a heavy chain variable region having the sequence described by SEQ ID NO: 77.

According to some embodiments, the isolated monoclonal antibody or fragment has at least 90% sequence identity with the light chain variable region described by SEQ ID NO: 79, or the light chain variable region sequence thereof. Including analog.

According to some embodiments, the analog of SEQ ID NO: 42 is a light chain variable region having the sequence described in SEQ ID NO: 79.

According to a specific embodiment, the isolated monoclonal antibody or fragment thereof is a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 77, and a SEQ ID NO. Includes light chain variable regions having sequences selected from the group consisting of: 42 and SEQ ID NO: 79, or their analogs with at least 90% sequence identity with light chain and / or heavy chain sequences.

According to some embodiments, the isolated monoclonal antibody or fragment thereof is a heavy chain variable region having the sequence set forth in SEQ ID NO: 34, and SEQ ID NO: 42, SEQ ID NO: 49, SEQ. Light chain variable region with the sequences set forth in ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 54; or light chain and / or heavy chain sequences. Includes those analogs with at least 90% sequence identity with. Each possibility represents a separate embodiment of the invention.

The present invention also surrounds an antibody or antibody fragment that allows high affinity binding to an epitope within a human PVR protein to which mAb 5B9 binds.

According to some embodiments, the isolated antibody or fragment thereof recognizes a human PVR with an affinity of ^{at least 10-8 M.} According to another embodiment, the antibody or antibody ^{fragment, 10 -8 M, 5x10 -9 M} , 10 -9 M, 5x10 -10 M, 10 -10 M, 5x10 -11 M, or above affinity By sex, it binds to human PVR. Each possibility represents a separate embodiment of the invention.

Analogs and derivatives of isolated mAb antibodies, as well as the antibody fragments described above, are also within the scope of the present invention. In some embodiments, a particular analog or isolated comprising at least one variable region described in a sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34, and 42. mAbs or fragments thereof are also within the scope of the present invention. In some embodiments, an isolated mAb or fragment thereof comprising at least one variable region described in a sequence selected from the group consisting of SEQ ID NO: 69, 71, 73, 75, 77, and 79. Is also within the scope of the present invention.

According to some embodiments, the analog of the antibody or antibody fragment has at least 90% sequence identity with the hypervariable region of the reference antibody sequence.

According to specific embodiments, the analog or derivative of the isolated antibody, or fragment thereof, is at least 91, 92, 93, 94, 95, 96, 97, 98, or 99 with the variable region of the reference antibody sequence. Has% sequence identity. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the antibody or antibody fragment according to the invention is a heavy chain variable region described in SEQ ID NO: 2 or SEQ ID NO: 69, or at least 95% sequence analogy to said sequence. Includes sex analog. According to other embodiments, the antibody or antibody fragment according to the invention has at least 95% sequence similarity to the heavy chain variable region described in SEQ ID NO: 18 or SEQ ID NO: 73, or to said sequence. Including analog with. According to other embodiments, the antibody or antibody fragment according to the invention has at least 95% sequence similarity to the heavy chain variable region described in SEQ ID NO: 34 or SEQ ID NO: 77, or to said sequence. Including analog with.

According to some embodiments, the antibody or antibody fragment is a light chain variable region described in SEQ ID NO: 10 or SEQ ID NO: 71, or an analog having at least 95% sequence similarity to said sequence. including. According to other embodiments, the antibody or antibody fragment comprises a light chain variable region described in SEQ ID NO: 26 or SEQ ID NO: 75, or an analog having at least 95% sequence similarity to said sequence. include. According to other embodiments, the antibody or antibody fragment comprises a light chain variable region described in SEQ ID NO: 42 or SEQ ID NO: 79, or an analog having at least 95% sequence similarity to said sequence. include.

According to some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, where: (i) the heavy chain comprises SEQ ID NO: 2 and the light chain comprises SEQ ID NO: 10; (Ii) Heavy chain comprises SEQ ID NO: 18, light chain comprises SEQ ID NO: 26; or (iii) heavy chain comprises SEQ ID NO: 34, light chain comprises SEQ ID NO: 42. .. Also included are analogs of antibodies or fragments that have at least 95% sequence similarity to the heavy or light chain.

According to other embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises SEQ ID NO: 69 and the light chain comprises SEQ ID NO: 71; ( ii) The heavy chain comprises SEQ ID NO: 73 and the light chain comprises SEQ ID NO: 75; or (iii) the heavy chain comprises SEQ ID NO: 77 and the light chain comprises SEQ ID NO: 79. Also included are analogs of antibodies or fragments that have at least 95% sequence similarity to the heavy or light chain.

According to some embodiments, the analog has at least 96, 97, 98, or 99% sequence identity with the variable region of the light or heavy chain of the antibody described above. According to some embodiments, the analog is one or more CDR sequences in the hypervariable region, ie, SEQ ID NO: 4, 6, 8, 12, 14, 16, 20, 22, 24, 28, 30, Substitution, deletion of only one amino acid into any one of the CDR sequences described in 32, 36, 38, 40, 44, 46, 48, 80, 81, 82, 83, 84, and 85. Or only include additions. Each possibility represents a separate embodiment of the invention. According to some embodiments, the amino acid substitution is a conservative substitution.

According to some embodiments, the antibody or antibody fragment is a light chain and light chain as defined above, in which 1, 2, 3, 4, or 5 amino acids are substituted, deleted, and / or added. Includes a hypervariable region (HVR) with heavy chains. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the antibody or antibody fragment comprises an HVR having a light chain and a heavy chain as defined above in which one amino acid is substituted. According to a specific embodiment, the antibody or antibody fragment comprises a CDR as defined above in which one amino acid is replaced. According to some specific embodiments, the antibody or antibody fragment comprises a light chain CDR2 as defined above in which one amino acid is substituted.

According to some specific embodiments, the antibody or antibody fragment comprises a light chain CDR2 having the sequence described in SEQ ID NO: 55 (WASSRHX), where X is A, R, D,. Selected from the group consisting of E, P and T. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the antibody or antibody fragment comprises a light chain CDR2 having a sequence selected from the group consisting of SEQ ID NO: 56-61.

According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a CDR set selected from the group consisting of:
i. A CDR set of 6 CDRs, where HC CDR1 is selected from GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84); HC CDR2 is EIFFPGSGRINFNEKFKG (SEQ ID NO: 38). Yes; HC CDR3 is TKIYGNSFDY (SEQ ID NO: 40); LC CDR1 is selected from KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85); LC CDR2 is WASSRHN. : 46), WASSRHA (SEQ ID NO: 56), WASSRHR (SEQ ID NO: 57), WASSRHD (SEQ ID NO: 58), WASSRHE (SEQ ID NO: 59), WASSRHP (SEQ ID NO: 60) and WASSRHT Selected from the group consisting of (SEQ ID NO: 61); and LC CDR3 is QQYSRYPLT (SEQ ID NO: 48).
ii. A CDR set of 6 CDRs, where the HC CDR1 sequence is selected from GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); HC CDR2 is EIHPDSSKINYTPSQ (SEQ ID NO: 6). ) And EIHPDSSKINYTPSQKD (SEQ ID NO: 81); HC CDR3 is selected from PDGNYNALDYW (SEQ ID NO: 8) and PDGNYNALDY (SEQ ID NO: 82); LC CDR1 is KASQDVGTAVT (SEQ ID NO: 82). The LC CDR2 is WASTRHT (SEQ ID NO: 14); and the LC CDR3 is QQYSRYPYT (SEQ ID NO: 16).
iii. A CDR set of 6 CDRs, where the HC CDR1 sequence is selected from GYTFTEYTMH (SEQ ID NO: 20) and EYTMH (SEQ ID NO: 83); HC CDR2 is GIDPNNGGTNYNQNFKG (SEQ ID NO: 22). The HC CDR3 is VIPLEY (SEQ ID NO: 24); the LC CDR1 is KASQNVYTNVA (SEQ ID NO: 28); the LC CDR2 is SASYRYR (SEQ ID NO: 30); and the LC CDR3 is. It is QQYNSYSPLA (SEQ ID NO: 32).

The present invention provides a monoclonal antibody that specifically binds human protein PVRs, or a binding fragment thereof, wherein the monoclonal antibody or fragment comprises a set of 6 CDR sequences, the set consisting of the following group. Selected:
i. SEQ ID NO. 4, 6, 8, 12, 14, and 16;
ii. SEQ ID NO. 20, 22, 24, 28, 30, and 32;
iii. SEQ ID NO. 36, 38, 40, 44, 46, and 48;
iv. SEQ ID NO. 36, 38, 40, 44, 55, and 48;
v. SEQ ID NO. 80, 81, 82, 12, 14, and 16;
vi. SEQ ID NO. 83, 22, 24, 28, 30, and 32;
vii. SEQ ID NO. 84, 38, 40, 85, 46 and 48; and viii. SEQ ID NO. 84, 38, 40, 85, 55, and 48.

The present invention also provides a monoclonal antibody and a binding fragment thereof comprising a heavy chain and a light chain, wherein the chain comprises a heavy chain variable region sequence and a light chain variable region sequence, and the set comprises the following group. Selected from:
i. SEQ ID NO: 2 and 10;
ii. SEQ ID NO: 69 and 71;
iii. SEQ ID NO: 18 and 26;
iv. SEQ ID NO: 73 and 75;
v. SEQ ID NOs: 34 and 42; and vi. SEQ ID NO: 77 and 79.

According to some embodiments, the antibody or antibody fragment can inhibit human PVR that binds to TIGIT expressed on T cells or NK cells.

According to a specific embodiment, the mAb is selected from the group consisting of chimeric antibodies and antibody fragments containing at least the antigen-binding portion of the antibody. According to a specific embodiment, the antibody is a chimeric antibody. According to other embodiments, the chimeric antibody comprises a human invariant region. According to other embodiments, the chimeric monoclonal antibody comprises a human IgG1 invariant region. According to specific embodiments, the antibody fragments are Fab, Fab', F (ab') ₂ , Fd, Fd', Fv, dAb, isolated CDR regions, single chain antibody (scab). , "Bispecific antibodies", and "linear antibodies". Each possibility represents a separate embodiment of the invention.

According to some embodiments, the antibody or antibody fragment comprises a framework sequence selected from the group consisting of mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2, human IgG3, and human IgG4. Each possibility represents a separate embodiment of the invention.

According to some embodiments, a complex comprising an antibody or fragment thereof as described above is provided.

According to some embodiments, the complex comprises a carrier protein.

The polynucleotide sequence encoding the monoclonal antibody has high affinity and specificity for the vector having these polynucleotide sequences and the host cell in addition to PVR, and is provided according to another aspect of the present invention.

According to some embodiments, a polynucleotide sequence encoding the amino acid sequences of the HC variable region and the LC variable region described above is provided.

According to some embodiments, the polynucleotide sequence encodes an antibody, antibody fragment, or chain capable of binding to an epitope within a human PVR protein, wherein the human PVR protein comprises (i) SEQ ID NO :. Monoclonal antibody with 2 heavy chain variable regions and light chain variable region of SEQ ID NO: 10 (identified herein as 4E5); (ii) heavy chain variable region of SEQ ID NO: 18 and SEQ ID NO : Monoclonal antibody with 26 light chain variable regions (identified herein as 7D4); or (iii) with heavy chain variable regions of SEQ ID NO: 34 and light chain variable regions of SEQ ID NO: 42. It binds to a monoclonal antibody (identified herein as 5B9).

According to some embodiments, the polynucleotide sequence encodes an antibody, antibody fragment, or strand that comprises the sequence described in SEQ ID NO: 2 or SEQ ID NO: 69. According to some embodiments, the polynucleotide sequence encodes an antibody, antibody fragment, or strand that comprises the sequence described in SEQ ID NO: 10 or SEQ ID NO: 71.

According to other embodiments, the polynucleotide sequence encodes an antibody, antibody fragment, or strand that comprises the sequence described in SEQ ID NO: 18 or SEQ ID NO: 73. According to additional embodiments, the polynucleotide sequence encodes an antibody, antibody fragment, or strand that comprises the sequence described in SEQ ID NO: 26 or SEQ ID NO: 75.

According to other embodiments, the polynucleotide sequence encodes an antibody, antibody fragment, or strand that comprises the sequence described in SEQ ID NO: 34 or SEQ ID NO: 77. According to additional embodiments, the polynucleotide sequence encodes an antibody, antibody fragment, or strand that comprises the sequence described in SEQ ID NO: 42 or SEQ ID NO: 79.

Also according to some embodiments, the polynucleotide sequence according to the invention encodes an antibody, antibody fragment, or chain containing the following six CDR sequences: (i) sequence: GFDFSRYW (SEQ ID NO:: 4) or heavy chain CDR1 with RYWMT (SEQ ID NO: 80), sequence: heavy chain CDR2 with EIHPDSSKINYTPSQ (SEQ ID NO: 6), sequence: heavy chain CDR3 with PDGNYNALDY (SEQ ID NO: 82), sequence : Light chain CDR1 with KASQDVGTAVT (SEQ ID NO: 12), sequence: Light chain CDR2 with WASTRHT (SEQ ID NO: 14), and sequence: Light chain CDR3 with QQYSRYPYT (SEQ ID NO: 16); (ii) ) Sequence: Heavy chain CDR1 with EYTMH (SEQ ID NO: 83), Sequence: Heavy chain CDR2 with GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), Sequence: Heavy chain CDR3 with VIPLEY (SEQ ID NO: 24), Sequence : Light chain CDR1 with KASQNVYTNVA (SEQ ID NO: 28), sequence: light chain CDR2 with SASYRYR (SEQ ID NO: 30), and sequence: light chain CDR3 with QQYNSYPLA (SEQ ID NO: 32); or ( iii) Sequence: Heavy chain CDR1 with SNYWIE (SEQ ID NO: 84), Sequence: Heavy chain CDR2 with EIFFGSGRINFNEKFKG (SEQ ID NO: 38), Sequence: Heavy chain CDR3 with TKIYGNSFDY (SEQ ID NO: 40), Sequence: light chain CDR1 with KASQDVGTAV (SEQ ID NO: 85), sequence: light chain CDR2 with WASSRHN (SEQ ID NO: 46), and sequence: light chain CDR3 with QQYSRYPLT (SEQ ID NO: 48).

According to some embodiments, the polynucleotide sequence defined above comprises a molecule selected from the group consisting of an antibody, an antibody fragment comprising at least an antigen-binding moiety, and an antibody complex comprising said antibody or antibody fragment. Code. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the polynucleotide sequence encoding the monoclonal antibody heavy chain variable region has the sequence described in SEQ ID NO: 1 or SEQ ID NO: 68, or at least 90% sequence identity. Includes that variant.

According to some embodiments, the polynucleotide sequence encoding the monoclonal antibody heavy chain variable region has the sequence described in SEQ ID NO: 17 or SEQ ID NO: 72, or at least 90% sequence identity. Includes that variant.

According to some embodiments, the polynucleotide sequence encoding the monoclonal antibody heavy chain variable region has the sequence described in SEQ ID NO: 33 or SEQ ID NO: 76, or at least 90% sequence identity. Includes that variant.

According to some embodiments, the polynucleotide sequence encoding the monoclonal antibody light chain variable region has the sequence described in SEQ ID NO: 9 or SEQ ID NO: 70, or at least 90% sequence identity. Includes that variant.

According to some embodiments, the polynucleotide sequence encoding the monoclonal antibody light chain variable region has the sequence described in SEQ ID NO: 25 or SEQ ID NO: 74, or at least 90% sequence identity. Includes that variant.

According to some embodiments, the polynucleotide sequence encoding the monoclonal antibody light chain variable region has the sequence described in SEQ ID NO: 41 or SEQ ID NO: 78, or at least 90% sequence identity. Includes that variant.

The present invention provides a polypeptide comprising at least one sequence encoded by at least one polynucleotide sequence disclosed above, according to some embodiments.

In a further aspect, the invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof according to the invention. According to some embodiments, the nucleic acid construct is a plasmid.

According to some embodiments, the plasmid comprises the polynucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 17, or SEQ ID NO: 33.

According to some embodiments, the plasmid comprises the polynucleotide sequence described in SEQ ID NO: 68, SEQ ID NO: 72, or SEQ ID NO: 76.

According to other embodiments, the plasmid comprises the polynucleotide sequence described in SEQ ID NO: 9, SEQ ID NO: 25, or SEQ ID NO: 41.

According to other embodiments, the plasmid comprises the polynucleotide sequence described in SEQ ID NO: 70, SEQ ID NO: 74 or SEQ ID NO: 78.

In yet another embodiment, the invention provides hybridoma cells capable of producing antibodies or antibody fragments containing the specific CDR sequences and / or specific heavy and light chain variable regions defined above. ..

According to some embodiments, hybridoma cells comprising at least one polynucleotide sequence disclosed above are provided.

According to some embodiments, the hybridoma can generate a monoclonal antibody containing the following six complementarity determining region (CDR) sequences: (i) sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT. Heavy chain CDR1 with (SEQ ID NO: 80), heavy chain CDR2 with sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3 with sequence: PDGNYNALDY (SEQ ID NO: 82), sequence: KASQDVGTAVT (SEQ) Light chain CDR1 with ID NO: 12), light chain CDR2 with sequence: WASTRHT (SEQ ID NO: 14), and light chain CDR3 with sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) sequence: EYTMH Heavy chain CDR1 with (SEQ ID NO: 83), heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), sequence: KASQNVYTNVA (SEQ). Light chain CDR1 with ID NO: 28), light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30), and sequence: light chain CDR3 with QQYNSYPLA (SEQ ID NO: 32); or (iii) sequence: Heavy chain CDR1 with SNYWIE (SEQ ID NO: 84), heavy chain CDR2 with sequence: EIFFGSGRINFNEKFKG (SEQ ID NO: 38), sequence: heavy chain CDR3 with TKIYGNSFDY (SEQ ID NO: 40), sequence: KASQDVGTAV Light chain CDR1 with SEQ ID NO: 85), light chain CDR2 with sequence: WASSRHN (SEQ ID NO: 46), and light chain CDR3 with sequence: QQYSRYPLT (SEQ ID NO: 48).

Those antibodies or fragments of the invention can be attached to cytotoxic, radioactive, or identifiable portions.

According to another embodiment, the present invention comprises an active ingredient recognizing a PVR having high affinity and specificity, at least one antibody, or an antibody fragment or complex thereof, and optionally at least one pharmaceutically acceptable additive. Provided are pharmaceutical compositions comprising a formant, a diluent, a salt, or a carrier.

According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof capable of binding to an epitope within a human PVR protein that binds to a monoclonal antibody selected from the group consisting of: (i). ) Antibodies identified herein as 4E5 (also referred to as PVR.07) having a heavy chain variable region of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO: 10; (ii) SEQ ID NO Antibodies identified herein as 7D4 (also referred to as PVR.01) with a heavy chain variable region of 18 and a light chain variable region of SEQ ID NO: 26; and (ii) SEQ ID NO: 34. An antibody identified herein as 5B9 (also referred to as PVR.09) having a heavy chain variable region and a light chain variable region of SEQ ID NO: 42.

According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or antibody fragment thereof comprising the following 6 CDRs: (i) sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO:: Heavy chain CDR1 with 80), heavy chain CDR2 with sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3 with sequence: PDGNYNALDY (SEQ ID NO: 82), sequence: KASQDVGTAVT (SEQ ID NO: 12) Light chain CDR1 with, sequence: WASTRHT (SEQ ID NO: 14) and light chain CDR2, and sequence: QQYSRYPYT (SEQ ID NO: 16) with light chain CDR3; Heavy chain CDR1 with 83), heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), sequence: KASQNVYTNVA (SEQ ID NO: 28) Light chain CDR1 with, sequence: light chain CDR2 with SASYRYR (SEQ ID NO: 30), and sequence: light chain CDR3 with QQYNSYPLA (SEQ ID NO: 32); or (iii) sequence: SNYWIE (SEQ ID NO: NO). Heavy chain CDR1 with: 84), heavy chain CDR2 with sequence: EIFFGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 with sequence: TKIYGNSFDY (SEQ ID NO: 40), sequence: KASQDVGTAV (SEQ ID NO: 85) , Light chain CDR1 with sequence: WASSRHN (SEQ ID NO: 46), and light chain CDR3 with sequence: QQYSRYPLT (SEQ ID NO: 48).

According to some embodiments, the pharmaceutical composition is from SEQ ID NO: 2, SEQ ID NO: 69, SEQ ID NO: 18, SEQ ID NO: 73, SEQ ID NO: 34 and SEQ ID NO: 77. Contains a monoclonal antibody or fragment thereof, which comprises a heavy chain variable region having a sequence selected from the group consisting of. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the pharmaceutical composition is from SEQ ID NO: 10, SEQ ID NO: 71, SEQ ID NO: 26, SEQ ID NO: 75, SEQ ID NO: 42 and SEQ ID NO: 79. Contains a monoclonal antibody or fragment thereof, which comprises a light chain variable region having a sequence selected from the group consisting of. Each possibility represents a separate embodiment of the invention.

According to a specific embodiment, the pharmaceutical composition comprises a heavy chain variable region having a sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 69, and in SEQ ID NO: 10 or SEQ ID NO: 71. Includes a monoclonal antibody or fragment thereof, which comprises a light chain variable region having the sequence described.

According to specific embodiments, the pharmaceutical composition comprises a heavy chain variable region having a sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 73, and in SEQ ID NO: 26 or DES ID NO: 75. Includes a monoclonal antibody or fragment thereof, which comprises a light chain variable region having the sequences described.

According to specific embodiments, the pharmaceutical composition comprises a heavy chain variable region having a sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 77, and in SEQ ID NO: 42 or SEQ ID NO: 79. Includes a monoclonal antibody or fragment thereof comprising a light chain variable region having the sequence described.

According to some embodiments, the pharmaceutical composition comprises a combination of at least two antibodies or antibody fragments that recognize human PVR.

According to other embodiments, the pharmaceutical composition is specific for one mAb or fragment that specifically binds to the PVR according to the invention and for different antigens such as cell receptors, tumor antigens, or immunomodulatory proteins. Includes one mAb or fragment that binds specifically.

Also, at least one antibody, antibody fragment, or antibody complex according to the invention for use in restoring NK cytotoxicity by inhibiting the binding of PVR to TIGIT expressed on NK cells. Also provided are pharmaceutical compositions containing.

According to some embodiments, the antibody, antibody fragment, or antibody complex can inhibit human PVR that binds TIGIT expressed on T cells.

According to some embodiments, the pharmaceutical compositions of the present invention are intended for use in cancer immunotherapy or in enhancing an immune response.

The cancer can be a cancer that expresses PVR. According to some embodiments, the cancer overexpresses PVR.

According to some embodiments of the invention, the cancer is a metastatic cancer. According to some embodiments, the pharmaceutical compositions of the present invention are for use to inhibit the formation or distribution of metastases in a subject or to reduce the total number of metastases.

According to some embodiments of the present invention, the cancers are melanoma, breast cancer, ovarian cancer, pancreatic cancer, colon cancer, colon cancer, cervical cancer, renal cancer, lung cancer, thyroid cancer, prostate cancer, brain cancer, It is selected from the group consisting of renal cancer, throat cancer, laryngeal cell tumor, bladder cancer, liver cancer, fibrosarcoma, endometrial cell carcinoma, glioblastoma, sarcoma, myeloid, leukemia, and lymphoma. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the cancer is a solid cancer. According to some specific embodiments, the solid cancer is selected from the group consisting of melanoma (skin), lung cancer, colon cancer, breast cancer, uterine cancer, and kidney cancer. According to specific embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, and liposarcoma.

According to other embodiments, the cancer is a blood cancer. According to some embodiments, the cancer of the blood is myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, multiple myeloma, or myelodysplastic syndrome. Each possibility represents a separate embodiment of the invention. According to a specific embodiment, the cancer is leukemia. According to a specific embodiment, the cancer is acute myeloid leukemia (AML).

According to some embodiments, the pharmaceutical compositions according to the invention are used in the treatment of viral infections.

According to some embodiments, the viral infection is caused by the virus that binds to the target cell via the PVR on the surface of the infected cell.

According to some embodiments, the pharmaceutical compositions of the present invention are used in the treatment of diseases or disorders associated with angiogenesis. According to certain embodiments, diseases or disorders associated with angiogenesis are selected from the group consisting of: cancer, cell proliferation of the eye (eye disease), retinal disorders and inflammatory diseases. Each possibility represents a separate embodiment of the invention.

According to yet another aspect, the invention provides a method of inhibiting the binding of human PVR to at least one ligand by using the monoclonal antibody or antibody fragment defined above.

According to a further aspect, the invention comprises the step of administering to said subject a therapeutically effective amount of a monoclonal antibody, antibody fragment or antibody complex as defined above, immunity in a subject in need thereof. Provide a method for enhancing the response.

According to yet another aspect, the present invention is capable of recognizing a human PVR with high affinity and specificity and inhibiting binding to its ligand, at least one antibody, antibody fragment or the like thereof. Provided is a step of treating a cancer, which comprises administering a therapeutically effective amount of a pharmaceutical composition, including a complex, to a subject in need thereof.

According to some embodiments, the antibody in the administered pharmaceutical composition is selected from the group consisting of: (i) the CDR sequence contained in the heavy chain variable region described in SEQ ID NO: 2 and the CDR sequence. , A monoclonal antibody comprising the CDR sequence contained in the light chain variable region described in SEQ ID NO: 10; (ii) the CDR sequence contained in the heavy chain variable region described in SEQ ID NO: 18 and the SEQ ID NO. : 26: Monoclonal antibody comprising the CDR sequence contained in the light chain variable region; or (iii) SEQ ID NO: 34 to the CDR sequence contained in the heavy chain variable region, and SEQ ID NO: 42. A monoclonal antibody comprising a CDR sequence, which is contained in the light chain variable region described.

According to some specific embodiments, the monoclonal antibody in the administered pharmaceutical composition comprises: heavy chain CDR1 consists of GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80). Contains sequences selected from the group; heavy chain CDR2 contains sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6); heavy chain CDR3 contains sequence: PDGNYNALDY (SEQ ID NO: 82); light chain CDR1 contains sequence: KASQDVGTAVT. (SEQ ID NO: 12); light chain CDR2 comprises sequence: WASTRHT (SEQ ID NO: 14); and light chain CDR3 comprises sequence: QQYSRYPYT (SEQ ID NO: 16). Each possibility represents a separate embodiment of the invention.

According to other specific embodiments, the monoclonal antibody in the administered pharmaceutical composition comprises: heavy chain CDR1 comprising sequence EYTMH (SEQ ID NO: 83); heavy chain CDR2 sequence: GIDPNNGGTNYNQNFKG (SEQ). ID NO: 22) is included; heavy chain CDR3 contains sequence: VIPLEY (SEQ ID NO: 24); light chain CDR1 contains sequence: KASQNVYTNVA (SEQ ID NO: 28); light chain CDR2 contains sequence: SASYRYR (SEQ ID NO: 28). Contains SEQ ID NO: 30); and light chain CDR3 comprises sequence: QQYNSYSPLA (SEQ ID NO: 32).

According to other specific embodiments, the monoclonal antibody in the administered pharmaceutical composition comprises: heavy chain CDR1 comprises sequence SNYWIE (SEQ ID NO: 84); heavy chain CDR2 contains sequence: EIFFGSGRINFNEKFKG (SEQ). ID NO: 38) is included; heavy chain CDR3 contains sequence: TKIYGNSFDY (SEQ ID NO: 40); light chain CDR1 contains sequence: KASQDVGTAV (SEQ ID NO: 85); light chain CDR2 is sequence: WASSRHN ( Contains SEQ ID NO: 46); and light chain CDR3 comprises sequence: QQYSRYPLT (SEQ ID NO: 48).

According to some embodiments of the invention, a therapeutically effective amount results in a reduction in tumor size or number of metastases in the subject.

According to some embodiments, the method of treating cancer comprises the step of administering or performing at least one additional anti-cancer treatment. According to certain embodiments, additional anti-cancer treatments are surgery, chemotherapy, radiation therapy or immunotherapy.

According to some embodiments, the method of treating cancer comprises administration of a monoclonal antibody that recognizes human PVR with high affinity and specificity, and an additional anti-cancer agent. According to some embodiments, additional anti-cancer agents are selected from the group consisting of: immunomodulators, activated lymphocyte cells, kinase inhibitors and chemotherapeutic agents.

According to other embodiments, the additional immunomodulatory agent is an antibody, antibody fragment or antibody complex that binds to an antigen other than human PVR.

According to some embodiments, the additional immunomodulatory agent is an antibody against an immune checkpoint molecule. According to some embodiments, additional immunomodulators include human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, cancer fetal antigen-related cell adhesion molecule 1 (CEACAM1), lymphocytes. For immune checkpoint molecules selected from the group consisting of activation gene 3 (LAG3), CD137, OX40 (also called CD134), killer cell immunoglobulin-like receptor (KIR), TIGIT, and any combination thereof. It is an antibody. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the anti-cancer agent is selected from the group consisting of: erbitax, cytarabine, fludarabine, fluorouracil, mercaptopurine, methoxantrone, thioguanine, gemcitabine, vincristine, vinblastin, vinolerubin, carmustine, lomustine, Chlormethine, cyclophosphamide, cisplatin, carboplatin, iphosphamide, mechlorethamine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorbisin, doxorubicin, idalbisin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide, and any A combination of them. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the anti-cancer agent is a dermal growth factor receptor (EGFR) inhibitor. According to some embodiments, the EGFR inhibitor is selected from the group consisting of: cetuximab (Elvitax®), panitumumab (Vectibix®) and necitumumab (Portrazza) ( Registered trademark)). According to some embodiments, the EGFR inhibitor is cetuximab (Elvitax®).

According to some embodiments of the invention, the subject is a human subject.

According to some embodiments of the invention, use further comprises the use of agents that down-regulate the activity or expression of immunocosuppressant receptors.

According to some embodiments of the invention, the immune cell is a T cell.

According to some embodiments of the invention, the immunoco-suppressive receptors are PD-1, TIGIT, DNAM-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55, LAIR1, SIGLEC10. And selected from the group consisting of 2B4. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, the present invention provides methods for regulating the function and / or activity of the immune system, including regulating the binding of PVR to TIGIT using antibodies according to the invention. ..

According to certain aspects, the invention provides a method of preventing or treating a viral infection of a virus that uses CD155 as an entry receptor in a subject in need, the method of which is described herein. It comprises the step of administering to the subject a therapeutically effective amount of the PVR monoclonal antibody. According to some embodiments, the virus is selected from the group consisting of: poliovirus, coxsackie virus, adenovirus and human immunodeficiency virus (HIV).

According to yet another aspect, the present invention provides a method for treating a disease or disorder associated with angiogenesis. According to certain embodiments, diseases or disorders associated with angiogenesis are selected from the group consisting of: cancer, proliferative disorders of ocular cells (diseases of the eye), retinal disorders and inflammatory disorders. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the method of treating cancer involves preventing or reducing the formation, growth, or nucleic acid of metastases in a subject by inhibiting angiogenesis.

According to certain embodiments, the present invention provides a method of diagnosing or predicting a subject's cancer or infectious disease, the method of which using at least one antibody described herein is the subject's organism. It comprises the step of determining the expression level of PVR in a scientific sample.

According to yet another aspect, the invention comprises a method of determining or quantifying the expression of PVRs, the method of contacting a biological sample with an antibody or antibody fragment, and the level of complex formation. The antibody or antibody fragment comprises a complementarity determining region (CDR) selected from the group consisting of: (i) Sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT ( Heavy chain CDR1 with SEQ ID NO: 80), heavy chain CDR2 with sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3 with sequence: PDGNYNALDY (SEQ ID NO: 82), sequence: KASQDVGTABT (SEQ ID). Light chain CDR1 with NO: 12), light chain CDR2 with sequence: WASTRHT (SEQ ID NO: 14), and light chain CDR3 with sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) sequence EYTMH ( Heavy chain CDR1 with SEQ ID NO: 83, heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), sequence: KASQNVYTNVA (SEQ ID NO). Light chain CDR1 with: 28), light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30), and light chain CDR3 with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) sequence SNYWIE. Heavy chain CDR1: sequence with (SEQ ID NO: 84) heavy chain CDR2 with sequence: EIFFGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 with sequence: TKIYGNSFDY (SEQ ID NO: 40), sequence: KASQDVGTAV (SEQ). Light chain CDR1 having ID NO: 85), light chain CDR2 having sequence: WASSRHN (SEQ ID NO: 46), and light chain CDR3 having sequence: QQYSRYPLT (SEQ ID NO: 48).

The determination and quantification methods may be performed in vitro or exvivo according to some embodiments, or may be used in diagnosing symptoms associated with the expression of PVR. Antibodies according to the invention may also be used to construct screening methods. For example, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), uses antibodies and methods known in the art to determine the level of secreted or cell-related polypeptides. Can be constructed to measure.

According to some embodiments, the method of detecting or quantifying the presence of PVR comprises the following steps:
i. The step of incubating a sample with an antibody specific for PVR, or an antibody fragment thereof containing at least an antigen-binding moiety;
ii. The step of detecting bound PVR using a detectable probe.
According to some embodiments, the method further comprises the following steps:
iii. A step of comparing the amount of (ii) with a standard curve obtained from a reference sample containing a known amount of PVR; and
iv. The step of calculating the amount of PVR in a standard curve sample.

According to some specific embodiments, the sample is a body fluid.

According to some embodiments, the method is performed in vitro or in Exvivo.

Kits for measuring PVR expression in biological samples are also provided containing at least one antibody or antibody fragment containing complementarity determining regions (CDRs) selected from the group consisting of: (i) Sequence: Heavy chain CDR1 with GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), sequence: heavy chain CDR2 with EIHPDSSKINYTPSQ (SEQ ID NO: 6), sequence: PDGNYNALDY (SEQ ID NO: 82). Heavy chain CDR3 with, sequence: light chain CDR1 with KASQDVGTAVT (SEQ ID NO: 12), sequence: light chain CDR2 with WASTRHT (SEQ ID NO: 14), and sequence: QQYSRYPYT (SEQ ID NO: 16). Light chain CDR3 with (ii) heavy chain CDR1 with sequence EYTMH (SEQ ID NO: 83), sequence: heavy chain CDR2 with GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), sequence: VIPLEY (SEQ ID NO: 24). Heavy chain CDR3 having, sequence: light chain CDR1 having KASQNVYTNVA (SEQ ID NO: 28), sequence: light chain CDR2 having SASYRYR (SEQ ID NO: 30), and sequence: QQYNSYPLA (SEQ ID NO: 32). Light chain CDR3 with; or heavy chain CDR1 with (iii) sequence SNYWIE (SEQ ID NO: 84), sequence: heavy chain CDR2 with EIFFGSGRINFNEKFKG (SEQ ID NO: 38), sequence: TKIYGNSFDY (SEQ ID NO: 40). , Heavy chain CDR3 with sequence: KASQDVGTAV (SEQ ID NO: 85), light chain CDR2 with sequence: WASSRHN (SEQ ID NO: 46), and sequence: QQYSRYPLT (SEQ ID NO: 48). ) With light chain CDR3.

According to certain aspects, the invention provides a kit for detecting cancer, the diagnostic kit comprising an antibody for the fragment thereof disclosed herein.

According to some embodiments, the present invention provides a method of diagnosing an immune-related or proliferative disease, assessing severity, or staging, wherein the method is an antibody according to the invention or a method thereof. It comprises a step of determining the expression or activity of PVR in a sample derived from a subject using a fragment or complex, and a step of comparing the expression or activity of PVR with the expression or activity of reference PVR. The reference amount may be obtained from a sample taken from a normal subject, from a sample taken from the same subject at different stages of the disease, or determined from clinical data of a large number of subjects. You may.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, since various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, detailed description and specific examples will be provided while showing preferred embodiments of the present invention. Please understand that it is given only as an example.

FIG. 1A is a graph showing the correlation between survival probability and (high or low) PVR mRNA expression levels (as shown). Correlation was measured for lung cancer (FIG. 1A). A dataset of mRNA expression was obtained from the GEO site as follows and bioprofiling. Analyzed using desite: GEO dataset ID: GSE321010 for lung cancer. FIG. 1B is a graph showing the correlation between survival probability and (high or low as shown) PVR mRNA expression levels. Correlation was measured for breast cancer (FIG. 1B). A dataset of mRNA expression was obtained from the GEO site as follows and bioprofiling. Analyzed using desite: GEO dataset ID: GSE25055 for breast cancer. FIG. 1C is a graph showing the correlation between survival probability and (high or low as shown) PVR mRNA expression levels. Correlation was measured for liposarcoma (Fig. 1C). A dataset of RNA expression was obtained from the GEO site as follows and bioprofiling. Analyzed using desite: GEO dataset ID: GSE30929 for liposarcoma. FIG. 2 is a schematic illustration of immunity and receptor expression on tumor cells. TIGIT relates to co-suppressive receptors on many immune cells (eg T cells); DNAM-1 (also referred to as CD226) relates to activated receptors on many immune cells (eg T cells); Fc Receptors relate to potently activated receptors that are expressed primarily in myeloid cells, including neutrophils and macrophages, as well as NK cells; PVR is expressed in many tumor cells, TIGIT (DNAM-). It relates to an inhibitory ligand for (which binds more weakly to 1); Nectin-2 relates to an activating ligand for DNAM-1 (marginal receptor by TIGIT), which is expressed in many tumor cells. Binding of anti-PVRs according to the invention has a dual effect: 1) enhance tumor cell death via Fc receptors; and 2) activate immune cells by blocking interaction with TIGIT. To increase. A-D of FIG. 3 is a graph for detecting FACS analysis of the four anti-PVR antibodies produced. It shows the efficacy of the antibody in blocking the direct binding of TIGIT-Fc to the tumor cell lineage. A in FIG. 3 illustrates a non-blocking anti-PVR antibody (also referred to as anti-PVR mAb antibody 2G3, hPVR.17). In FIG. 3, three of the antibodies produced, namely 5B9 (also called hPVR.09), 7D4 (also called hPVR.01) and 4E5 (also called hPVR.07), are TIGIT-, respectively. It is shown to be an anti-PVR blocking mAb as more indicated by blocking Ig binding. Hybridoma soup (5 μl per well) was added to HepG2 cells. TIGIT-Fc was used at 2 μg / well to a final concentration of 20 μg / ml, fluorescently labeled anti-FcAb was added, and then the level of cell-bound TIGIT was measured by FACS. The filled histogram shows background staining with anti-Fc reagents. FIG. 4 shows how blocking the PVR-TIGIT interaction with the anti-PVR mAb antibody 7D4 (also called hPVR.01) enhances NK cell killing in the human cell line MDA-MB-231 (breast cancer). It is a graph which shows. Specific death is calculated based on the secretion of [35S] --methionine from target cells. The control (Ctrl) is an unrelated mouse IgG. P value = 0.0056. FIG. 5 is a graph showing that blocking the PVR-TIGIT interaction using anti-PVR mAb4E5 (also called hPVR.07) enhances NK cell killing in human cancer cell line HepG2 (hepatocellular carcinoma). Death of HepG2 without -mAb; Death of HepG2 with anti-PVR 4E5-mouse anti-PVR 4E5mIgG1 (no activation of human Fc receptors); Death of HepG2 with anti-PVR 4e5hIgG1-anti-PVR 4E5-hIgG1 (human) Fc receptor activation), erbitux (PC) -positive control mAb erbitux (anti-EGFR). All mAbs were used at 10 μg / ml. P values: anti-PVR 4E5-0.04, anti-PVR 4e5hIgG1-0.000746, and erbitux (positive control) -0.003219. FIG. 6A is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Melanoma cells (FIGS. 6A-E) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6B is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Melanoma cells (FIGS. 6A-E) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6C is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Melanoma cells (FIGS. 6A-E) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also called hPVR.07). FIG. 6D is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Melanoma cells (FIGS. 6A-E) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also called hPVR.07). FIG. 6E is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Melanoma cells (FIGS. 6A-E) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also called hPVR.07). FIG. 6F is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Breast cancer cells (Fig. 6F-H) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6G is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Breast cancer cells (Fig. 6F-H) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6H is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Breast cancer cells (Fig. 6F-H) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6I is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Colon cells (FIG. 6I) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6J is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Kidney cells (Fig. 6J) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6K is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Lung cancer cells (Fig. 6K) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6L is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Prostate cancer cells (FIG. 6L) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6M is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Brain tumor cells (Fig. 6M) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6N is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Hepatocellular carcinoma cells (Fig. 6NO) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). FIG. 6O is a graph illustrating that human tumor cell lines express PVR and Nectin-2. Hepatocellular carcinoma cells (Fig. 6NO) express PVR and Nectin-2. mAbs were used at 0.2 μg / well: commercially available anti-Nectin-2 mAbs and anti-PVR mAbs 4E5 (also referred to as hPVR.07). A-C of FIG. 7 is a graph of FACS analysis showing that PVR is the predominant TIGIT ligand. A in FIG. 7 illustrates that HepG2 cells (human hepatocellular carcinoma cells) express PVR and Nectin-2. B in FIG. 7 shows that purified anti-PVR mAb 4E5 (also called hPVR.07) (0.15 μg) is almost completely (97%) despite the fact that these cells also express Nectin-2. Illustrates the blocking of super) TIGIT-Ig (2 μg / ml) binding. C in FIG. 7 illustrates that CHO cells express high levels of hnectin-2. D in FIG. 7 shows the lack of staining of the same CHO cells as C in FIG. 7 by all PVR mAbs, which means that there is no direct recognition of Nectin-2 by anti-PVR mAbs, and therefore the figure. The blockade of TIGIT binding seen in B of 7 cannot be explained by blockade of Nectin-2, but rather indicates that it is the result of direct PVR blockade. FIG. 8A-C illustrates all similar binding potency of anti-PVR mAbs to human PVR. All three blocking clones produce similar (less than 10% difference) binding to both endogenous PVR HepG2 cells (FIG. 8A) and overexpressed hPVR B16-hPVR cells (FIG. 8B). Binding was also examined using a Vero cell line (FIG. 8C) from an African green monkey expressing a PVR protein with 93% similarity to human PVR. In this case, the different Abs showed different staining intensities. 0.2 μg of each mAb was used in all cases. FIG. 8A-C illustrates all similar binding potency of anti-PVR mAbs to human PVR. All three blocking clones produce similar (less than 10% difference) binding to both endogenous PVR HepG2 cells (FIG. 8A) and overexpressed hPVR B16-hPVR cells (FIG. 8B). Binding was also examined using a Vero cell line (FIG. 8C) from an African green monkey expressing a PVR protein with 93% similarity to human PVR. In this case, the different Abs showed different staining intensities. 0.2 μg of each mAb was used in all cases. FIG. 8A-C illustrates all similar binding potency of anti-PVR mAbs to human PVR. All three blocking clones produce similar (less than 10% difference) binding to both endogenous PVR HepG2 cells (FIG. 8A) and overexpressed hPVR B16-hPVR cells (FIG. 8B). Binding was also examined using a Vero cell line (FIG. 8C) from an African green monkey expressing a PVR protein with 93% similarity to human PVR. In this case, the different Abs showed different staining intensities. 0.2 μg of each mAb was used in all cases. FIG. 9 is a graph showing that the anti-PVR antibody of the present invention does not recognize canine PVR. Human TIGIT-Fc (10 μg / ml) strongly cross-reacts and binds to the canine MDCK cell line. Neither PVR mAb was able to bind to these cells, suggesting that it did not recognize canine PVR. FIG. 10A shows that Nectin-2 is preferentially bound by DNAM-1 and not by TIGIT. Cells expressing either PVR or Nectin-2 were stained using the indicated antibody concentrations. FIG. 10A illustrates the binding of TIGIT-Fc to Nectin-2 expressing cells. FIG. 10B shows that Nectin-2 is preferentially bound by DNAM-1 and not by TIGIT. Cells expressing either PVR or Nectin-2 were stained using the indicated antibody concentrations. FIG. 10B illustrates the binding of DNAM-FC to Nectin-2 expressing cells. FIG. 10C shows that Nectin-2 is preferentially bound by DNAM-1 and not by TIGIT. Cells expressing either PVR or Nectin-2 were stained using the indicated antibody concentrations. FIG. 10C illustrates the binding of TIGIT to PVR-expressing cells. FIG. 10D shows that Nectin-2 is preferentially bound by DNAM-1 and not by TIGIT. Cells expressing either PVR or Nectin-2 were stained using the indicated antibody concentrations. FIG. 10D illustrates the binding of DNAM-1 to PVR-expressing cells. FIG. 11 shows the effect of anti-PVR antibody on T cell proliferation. Human PBMCs were CFSE labeled and incubated with target cells in the presence of the indicated antibodies. Results are presented as a factor of increased proliferation compared to controls. The results are for 7 pooled experiments with a total of 10 healthy donors. P value: 0.000241 for mAb 4E5, 1.96E- ⁰⁵ for mAb 5B9, 0.016303 for anti-PD-1, 0.000171 for anti-CTLA4, and 0.008171 for 4E5hIgG1. FIG. 12 shows the effect of the combined use of anti-PVR antibody and other antibodies on T cell proliferation. Human PBMCs were CFSE labeled and incubated with target cells in the presence of the indicated antibody combinations. Results are presented as a factor of increased proliferation compared to controls. The results are 7 independent experiments with 12 healthy donors. P value: anti-PD1 + anti-CTLA-47.54E- ⁰³ , anti-PD-1 + 4E5-7.02E- ⁰² , anti-PD-1 + 5B9-1.11E- ⁰⁴ , anti-CTLA4 + 4E5-1.37E- ⁰³ , anti-CTLA4 + 5B9-5. 47E- ⁰⁶ . FIG. 13 shows the specific effect of anti-PVR antibody on the proliferation of CD8 T cells. Human PBMCs were CFSE labeled and incubated with target cells (A549) in the presence of the indicated antibodies. CD8 positive cells were tabulated by FACS after 9-12 days of culture. Results are presented as a factor of increased proliferation compared to controls. The results are for two independent experiments with healthy PBMC donors. FIG. 14 shows the ratio of CD8 cells to CD4 cells after induction with different antibodies. Human PBMCs were CFSE labeled and incubated with target cells (A549) in the presence of the indicated antibodies. Cells were aggregated after 9-12 days of culture. The results are for two pooled experiments with a total of two healthy donors. FIG. 15 shows the effect of the anti-PVR antibody on NK degranulation. Human NK cells were incubated with MDA-MB-231 cells (three-type negative breast cancer cell line) in the presence of the indicated antibody. The results are representative of seven independent experiments performed on five different healthy NK cell donors. P value: PVR4E5-0.005063, PVR5B9-0.00374, PVR7D4-0.019448, PVR4E5-hIgG1-2.03E- ⁰⁵ , PVR5B9-hIgG1-1.45E- ⁰⁵ , PVR7D4-hIgG1-5.8E- ⁰⁵ .. FIG. 16A shows the effect of anti-PVR antibodies on tumor cell survival in the absence of immune cells. Survival of A549 cells (FIG. 16A) was examined using the MTT cell survival assay for 24 hours in the presence of the indicated mAbs of 50 micrograms / ml. Significance is calculated by the single tailed Student's T-test, ^* <0.05, ^** <0.03, and ^{*** <0.02.} FIG. 16B shows the effect of anti-PVR antibodies on tumor cell survival in the absence of immune cells. Survival of U373 cells (FIG. 16B) was examined using the MTT cell survival assay for 24 hours in the presence of the indicated mAbs of 50 micrograms / ml. Significance is calculated by the single tailed Student's T-test, ^* <0.05, ^** <0.03, and ^{*** <0.02.} FIG. 16C shows the effect of anti-PVR antibodies on tumor cell survival in the absence of immune cells. Survival of HCT116 cells (FIG. 16C) was examined using the MTT cell survival assay for 24 hours in the presence of the indicated mAbs of 50 micrograms / ml. Significance is calculated by the single tailed Student's T-test, ^* <0.05, ^** <0.03, and ^{*** <0.02.} FIG. 16D shows the effect of anti-PVR antibodies on tumor cell survival in the absence of immune cells. Survival of Mel-624 (FIG. 16D) was examined using the MTT cell survival assay for 24 hours in the presence of the indicated mAbs of 50 micrograms / ml. Significance is calculated by the single tailed Student's T-test, ^* <0.05, ^** <0.03, and ^{*** <0.02.}

The present invention provides monoclonal antibodies specific for the human poliovirus receptor (PVR). The present invention also provides the production and use of mAbs as therapeutic agents. In particular, the mAbs of the present invention can be used alone or in combination with other agents to restore and enhance the antitumor killing activity of immune cells and as diagnostic reagents.

As used herein, the term "antigen" refers to a molecule or portion of a molecule that can trigger antibody formation and is specifically bound by the antibody. The antigen may have one or more epitopes. The specific binding mentioned above indicates that the antigen reacts with its corresponding antibody and not with many other antibodies that can be induced by other antigens in a highly selective manner. Means that. The antigen according to some embodiments of the present invention is a PVR protein.

As used herein, the term "PVR" refers to a poliovirus receptor, also known as CD155 (cluster of differentiation 155). A PVR is a transmembrane glycoprotein with an N-terminal signal sequence, three extracellular immunoglobulin (Ig) -like domains, a transmembrane domain, and a cytoplasmic terminal. It has a molecular size of about 80 kDa and a structure consisting of three Ig-like domains, specifically the outermost V-like domain followed by two C2-like domains. Exemplary PVRs according to the invention are described by GenBank Accession Numbers: NP_001192940.1, NP_0011929241.1, NP_0011929242.2 and NP_006496.4. These poliovirus receptors share a sequence of extracellular domains and can therefore be targeted by the affinity binding moieties of the invention.

As used herein, the term "antigen determinant" or "epitope" refers to a region of an antigen molecule that specifically reacts with a particular antibody. Peptide sequences derived from epitopes, either alone or in combination with carrier moieties, can be used to immunize animals and produce additional polyclonal or monoclonal antibodies by applying methods known in the art. Can be done. The isolated peptide derived from the epitope may be used diagnostically to detect the antibody.

Affinity is described in Surface Plasmon Resonance (SPR) (Scarano S, Mascini M, Turner AP, Minunni M. Surface resonance resonance imaging for affiliity-based biosensors. It should be noted that it can be quantified using the method and can be calculated, for example, using the dissociation constant Kd so that the lower Kd reflects the higher affinity.

Antibodies or fragments thereof according to the present invention bind to epitopes of hPVR. Specifically, the antibody binds to an epitope within amino acids 1-343 of PVR described in NP_006496.4.

An antibody or immunoglobulin contains a disulfide bond and two heavy chains attached to each other by two light chains, each light chain being linked to each heavy chain by a "Y" -shaped disulfide bond. Proteolytic digestion of antibodies results in Fv (variable fragment) and Fc (fragment crystalline) domains. The antigen-binding domain Fab comprises a region where the polypeptide sequence changes. The term F (ab') 2 represents two Fab'arms linked together by a disulfide bond.
The central axis of the antibody is called the Fc fragment. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at the other end, the light chain variable domain aligns with the heavy chain variable domain, and the light chain constant domain is heavy chain (CL). It is aligned with the first constant domain of CH1). The variable domains of each pair of light and heavy chains form an antigen-binding site. The domains on the light and heavy chains have the same general structure, each domain containing four framework regions, and the sequences of the four framework regions are relatively conserved and complementarity determining regions (CDR1). It is linked by three hypervariable domains known as -3). These regions contribute to the specificity and affinity of the antigen binding site.

CDR identification or determination from a given heavy or light chain variable sequence is typically made using one of the few methods known in the art. For example, such determinations include Kabat (Wu TT and Kabat EA, J Exp Med, 1970; 132: 211-50) and IMGT (Lefranc MP, et al., Dev Comp Immunol, 2003). , 27: 55-77).

When the term "CDR with sequence" or similar term is used, this also includes options where the CDR contains a particular sequence and where the CDR consists of a particular sequence.

The antigen specificity of an antibody is based on hypervariable regions (HVRs), the unique CDR sequences of both light and heavy chains that together form an antigen binding site.

The heavy chain isotype (gamma, alpha, delta, epsilon or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either of two isotypes (kappa kappa or lambda λ). Both isotopes are found in all antibody classes.

The term "antibody" is used in the broadest sense to mean a monoclonal antibody of sufficient length (full-length or intact) to exhibit the desired biological activity, i.e. binding to human PVR. Includes), polyclonal antibodies, multivalent antibodies, and antibody fragments.

Antibodies according to the invention include complete antibodies such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof, i.e. fragments such as Fab or F (ab') 2 fragments. Single chain antibodies also fall within the scope of the invention.

<Antibody fragment>
An "antibody fragment" comprises only a portion of a complete antibody, generally comprises the antigen binding site of the complete antibody, and thus retains the ability to bind the antigen. Examples of antibody fragments included by this definition include: (i) FAb fragments with VL, CL, VH and CH1 domains; (ii) Fab'fragments, i.e. one or more cysteines at the C-terminal of the CH1 domain. FAb fragment with residues; (iii) Fd fragment with VH and CH1 domains; (iv) VH and CH1 domains, and Fd'fragment with one or more cysteine residues at the C-terminal of the CH1 domain; (v) ) Fv fragment having a single arm VL and VH domain of antibody; (vi) dAb fragment consisting of VH domain (Ward et al., Nature 1989, 341, 544-546); (vi) isolated CDR region; (Viii) F (ab') 2 fragments, i.e. divalent fragments containing two Fab' fragments linked by disulfide cross-linking in the hinge region; (ix) single chain antibody molecule (eg, single chain Fv; scFv) ( Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. (USA) 1988, 85, 5879-5883); (x) Light chain in the same polypeptide chain. A "bispecific antibody" with two antigen binding sites, including a heavy chain variable domain (VH) bound to a variable domain (VL) (eg, EP 404,097; WO 93/11161; and Hollinger. et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) A pair of tandem Fd forming a pair of antigen-binding regions with a complementary light chain polypeptide. A "linear antibody" containing a segment (VH-CH1-VH-CH1) (Zapata et al. Protein Eng., 1995, 8, 1057-1062; and US Pat. No. 5,641,870).

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been obtained by protein digestion of complete antibodies (eg, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992), and Brennan et al., Science, 2). 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, the Fab'-SH fragment can be recovered directly from E. coli and chemically bound to form the F (ab') 2 fragment (Carter et al., Bio / Technology 10: 163-). 167 (1992)). According to another approach, the F (ab') 2 fragment can be isolated directly from the culture of recombinant host cells. Other techniques for the production of antibody fragments will be apparent to those of skill in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv).

Single chain antibodies are single chain synthetics that have the ability to bind antigens and contain amino acid sequences that are homologous or similar to the light and heavy chain variable regions of immunoglobulins, namely chained VH-VL or single chain Fv (scFv). It can be a polypeptide. Techniques for producing single-stranded antibodies (US Pat. No. 4,946,778) can be applied to produce single-stranded antibodies against PVR.

As used herein, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are naturally present in minor amounts. It is the same except for the mutations that can occur. Monoclonal antibodies are highly specific and target a single antigen. Moreover, each monoclonal antibody targets a single determinant in the antigen, as opposed to polyclonal antibody preparations that contain different antibodies that typically target different determinants (epitopes). The modifier "monoclonal" is not construed as requiring the production of an antibody by any particular method. mAbs will be obtained by methods known to those of skill in the art. For example, the monoclonal antibody used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (Kohler et al., Nature 1975, 256, 495), or the recombinant DNA method (eg, USA). It may be made according to Patent No. 4,816,567). Monoclonal antibodies are further described, for example, in Clackson et al. (Clackson et al., Nature 1991, 352, 624-628) or Marks et al. (Marks et al., J. Mol. Biol., 1991, 222: 581-597). Techniques may be used to isolate from the phage antibody library.

Design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies by rapid identification and cloning of functional variable heavy chain (VH) and variable light chain (VL) genes, and in recombinant bacteria. The design and cloning of synthetic DNA sequences optimized for expression is described in Fields et al. (Fields et at. 2013, 8 (6): 1125-48).

The mAb of the present invention may be any immunoglobulin class including IgG, IgM, IgE, IgA and IgD. Hybridomas that produce mAbs can be cultured in vitro or in vivo. High titers of mAbs can be obtained by in vivo production, in which cells from individual hybridomas are peritoneally stimulated in Balb / c mice to produce ascites containing high concentrations of the desired mAB. It is administered internally. mAbs may be purified from such ascites or culture supernatant using methods well known to those of skill in the art.

Further understood are anti-idiotype antibodies that specifically immune react to the hypervariable regions of the antibodies of the invention.

The present invention provides a monoclonal antibody or antibody fragment comprising an antigen binding domain (ABD) comprising three CDRs of a light chain and three CDRs of a heavy chain, wherein the ABD is at least 90% of the ABD of the monoclonal mouse antibody. Has sequence identity or similarity: (i) variable heavy chain containing amino acid sequence SEQ ID NO: 69, and amino acid sequence SEQ ID NO: 71 (specified herein as 4E5 or hPVR.07). Variable light chain comprising; (ii) Variable heavy chain comprising amino acid sequence SEQ ID NO: 73, and variable light chain comprising amino acid sequence SEQ ID NO: 75 (specified herein as 7D4 or hPVR.01). ABD comprising; or (iii) a variable heavy chain comprising the amino acid sequence SEQ ID NO: 77, and a variable light chain comprising the amino acid sequence SEQ ID NO: 79 (specified herein as 5B9 or hPVR.09). Is. Such antibodies have at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, at least 99% sequence identity or similarity to the corresponding ABD of 4E5, 7D4 or 5B9. , Or may have an ABD domain with 100% sequence identity.

Sequence identity is the amount of amino acids or nucleotides that exactly match between two different sequences. Sequence similarity makes it possible to determine conservative substitutions of amino acids as identical amino acids.

The present invention further provides conservative amino acid variants of the antibody molecule according to the invention. Further variants of the invention can be made that preserve the overall molecular structure of the encoded protein. Considering the properties of the individual amino acids, including the disclosed protein products, some reasonable substitutions will be recognized by those of skill in the art. Amino acid substitutions, or "conservative substitutions," may be made, for example, on the basis of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues contained. As used herein, the term "antibody analog" refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.

As used herein, the term "antibody variant" refers to any molecule, including the antibodies of the invention. For example, a fusion protein in which an antibody or its antigen-binding fragment is linked to another chemical component is considered an antibody variant.

Antibody sequence analogs and variants are also within the scope of this application. These include, but are not limited to, conservative and non-conservative substitutions, insertions and deletions of amino acids in the sequence. Such modifications and resulting antibody analogs or variants are within the scope of the invention if they result in or further improve binding of the antibody to human PVR.

Conservative substitutions of amino acids as known to those of skill in the art are within the scope of the present invention. Conservative substitution of an amino acid involves replacing one amino acid with another amino acid having the same type of functional group or side chain, such as an aliphatic, aromatic, positive or negative charge. These substitutions can enhance oral bioavailability, permeability, and targeting of specific cell populations and immunogenicity. Those skilled in the art will alter, add, or delete a single amino acid or a few percent of the amino acids in the encoded sequence, individual substitutions, deletions, or additions to the peptide or polypeptide or protein sequence, by alteration. You will recognize that it is a "conservatively modified variant" that results in the substitution of amino acids with chemically similar amino acids. A list of conservative substitutions that provide functionally similar amino acids is well known in the art. For example, according to a list known in the art, each of the following six groups contains amino acids that are conservative substitutions for each other:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), glutamic acid (E);
3) Asparagine (N), glutamine (Q);
4) Arginine (R), Lysine (K);
5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

It should be emphasized that the sequence of the mutant strand is determined by sequencing methods using specific primers. Due to technical issues and different primers, different sequencing methods used on the same sequence can result in slightly different sequences, especially at the ends of the sequence. Therefore, variants with different anti-PVR variable chain sequences are identified according to the present invention.

As used herein, the terms "molecule having an antigen-binding portion of an antibody" and "antigen-binding fragment" are not only isotypes and complete immunoglobulin molecules produced by any animal cell line or microorganism. FAb fragments, Fab'fragments, F (ab') 2 fragments, antigen-binding reactive fragments containing variable portions of their heavy and / or light chains, Fab small antibodies (eg, WO 93/15210), but not limited to , U.S. Patent Application No. 08 / 256,790, International Publication No. 96/13583, U.S. Patent Application No. 08 / 817,788, International Publication No. 96/37621, U.S. Patent Application No. 08/999,554), and the like. It is intended to include single chain antibodies incorporating such reactive fragments, as well as any other type of molecule into which such reactive fragments are physically inserted. Such molecules can be provided by known techniques, including but not limited to enzymatic cleavage, peptide synthesis or recombination techniques.

Monoclonal antibodies herein are one of a heavy chain and / or a light chain to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, as long as it exhibits the desired biological activity. The rest of the chain is identical or homologous to the corresponding sequence of an antibody that is identical or homologous in part and, on the other hand, is derived from another species or belongs to another antibody class or subclass, which is a fragment of such an antibody. Also includes "chimeric" antibodies (US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 [1984]). In addition, complementarity determining region (CDR) transplantation may be performed to alter specific properties of an antibody molecule, including affinity or specificity. Non-limiting examples of CDR transplantation are disclosed in US Pat. No. 5,225,539.

A chimeric antibody is a molecule derived from an animal species in which various parts of the molecule are different, such as those having a variable region derived from mouse mAb and a constant region of human immunoglobulin. Antibodies having variable region framework residues that are substantially derived from human antibodies (called receptor antibodies) and CDRs that are substantially derived from mouse antibodies (called donor antibodies) are also referred to as humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in administration and increase yield in production, eg, mouse mAbs have higher yields from hybridomas, but higher immunogenicity is in humans. If a human / mouse chimeric mAb is used. Chimeric antibodies and methods for producing them are known in the art (eg, PCT Patent Application International Publication No. 86/01533, International Publication No. 97/02671, International Publication No. 90/07861, International Publication No. 92/22653, and US Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101, and 5,225,539).

According to some specific embodiments, the monoclonal antibody is a chimeric monoclonal antibody.

According to some embodiments, the chimeric antibody comprises a constant region of human origin.

According to some embodiments, the human constant region of the chimeric antibody is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

According to certain embodiments, the chimeric monoclonal antibody or fragment thereof comprises a constant region subclass of the human IgG1 subtype.

According to certain embodiments, a chimeric monoclonal antibody that recognizes PVR is provided and comprises a set of 6 CDRs selected from the group consisting of: (i) SEQ ID NO: 4 or 80, 6 Or 81, 8 or 82, 12, 14, and 16; (iii) SEQ ID NO: 20 or 83, 22, 24, 28, 30, and 32; and (iii) SEQ ID NO: 36 or 84, 38, 40, 44 or 85, 46, and 48.

<Pharmacology>
In pharmaceutical and pharmaceutical formulations, the active agent is preferably utilized with one or more pharmaceutically acceptable carriers and optionally any other therapeutic ingredient. The carrier should be pharmaceutically acceptable in the sense that it is compatible with the other ingredients of the formulation and is not overly harmful to its recipients. The active agent is provided in an amount effective to achieve the desired pharmaceutical effect as described above, and in an amount appropriate to achieve the desired exposure.

Typically, the antibodies and fragments of the invention and their complexes containing an antigen-binding portion of the antibody or containing another polypeptide containing a peptide mimetic are suspended in sterile saline for therapeutic use. It becomes muddy. Alternatively, the pharmaceutical composition may be dispensed to control the release of the active ingredient (a molecule containing an antigen-binding portion of an antibody) or to prolong its presence in the patient's organ system. Many suitable drug delivery systems are known, including, for example, implantable drug release systems, hydrogels, hydroxymethyl celluloses, microcapsules, liposomes, microemulsions, microspheres and the like. The controlled release formulation can be prepared according to the present invention through the use of polymers for complexing or adsorbing molecules. For example, biocompatible polymers include a matrix of poly (ethylene-co-vinyl acetate) and a matrix of polyanamic copolymers of stearic acid dimer and sebacic acid. The rate of release of a molecule, ie, an antibody or antibody fragment, according to the invention from such a matrix depends on the molecular weight of the molecule, the amount of molecules in the matrix, and the size of the dispersed particles.

The pharmaceutical compositions of the present invention may be administered by any suitable method, such as oral, topical, intranasal, subcutaneous, intramuscular, intravenous, intraarterial, intra-articular, intra-injury, intratumoral, or parenteral. .. Intravenous (iv) administration is usually used to deliver the antibody.

The therapeutically effective amounts of the molecules according to the invention are, among other things, the schedule of administration, the unit dose of the molecule administered, whether the molecule was co-administered with other therapeutic agents, the patient's immune status and health status, and the molecule administered. It will be apparent to the usual practitioner that it depends on the therapeutic activity of the drug, its persistence in the blood circulation, and the judgment of the treating physician.

As used herein, the term "therapeutically effective amount" refers to the amount of drug effective in treating a disease or disorder in a mammal. In the case of cancer, therapeutically effective amounts of the drug reduce the number of cancer cells; reduce tumor size; inhibit the invasion of cancer cells into peripheral organs (ie, delay to some extent, and preferably stop). Inhibits tumor metastasis (ie, delays to some extent, and preferably stops); inhibits tumor growth to some extent; and / or can alleviate one or more symptoms associated with injury to some extent. It can be cell proliferation inhibitory and / or cytotoxic to the extent that the drug can prevent the growth of existing cancer cells and / or kill them. For cancer treatment, in vivo efficacy is measured, for example, by assessing survival, time of disease progression (TTP), response rate (RR), duration of response, and / or quality of life assessment. It is possible.

Cancers that can be modified with respect to the treatment according to the invention include, but are not limited to: cell tumors, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More detailed examples of such cancers are squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (gastrointestinal). Cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colon cancer, endometrial cancer or uterine body cancer, salivary adenocarcinoma, kidney cancer , Liver cancer, prostate cancer, genital cancer, thyroid cancer, liver cancer and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade / follicular non-hodgkin lymphoma (NHL)); small lymphocytic (SL) ) NHL; Medium-grade / follicular NHL; Medium-grade diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cleaved cell NHL; giant mass NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrem macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia Leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal defect growth associated with mammary plaques, edema (such as those associated with brain tumors) and Maegus syndrome. Preferably, the cancers are breast cancer, colon cancer, rectal cancer, non-small cell lung cancer, non-hodgkin lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, capogiosarcoma, cartinoid cancer, head and neck. It is selected from the group consisting of partial cancer, melanoma, ovarian cancer, mesenteric tumor, and multiple myeloma. Cancerous diseases that can be modified with respect to the treatment of the present invention include metastatic cancer.

According to other embodiments, the pharmaceutical compositions according to the invention are for use in the treatment of cancers characterized by PVR overexpression. PVR overexpression associated with the type of cancer can be identified using known databases such as The Cancer Genome Atlas (TCGA). According to certain embodiments, the cancers are adrenocortical carcinoma (ACC), clear cell carcinoma (KICH), hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD, READ), Pancreatic adenocarcinoma (PAAD)), chromium-affinity cell tumor & paraganglioma (PCPG), papillary kidney cancer (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate cancer (PRAD) , Uterine Endometrial Carcinoma (UCEC), Cervical Cancer (CESC), Cutaneous Adenocarcinoma (SKCM), Mesoderma (MESO), Urinary Adenocarcinoma (BLCA), Clear Cell Adenocarcinoma (KIRC), Lung Spinous cell carcinoma (LUSC), uterine carcinoma sarcoma (UCS), sarcoma (SARC), ovarian serous cystadenocarcinoma (OV), papillary thyroid cancer (THCA), polymorphonuar glioblastoma (GBM), breast cancer ( It is selected from the group consisting of BRCA), low-grade glioma (LGG), and diffuse large-cell B-cell lymphoma (DLBC). The individual possibilities represent separate embodiments of the invention.

As an active ingredient, the molecule of the present invention is, as is well known, dissolved, dispersed or mixed in an excipient that is pharmaceutically acceptable and has an affinity for the active ingredient. Suitable excipients are, for example, water, saline solution, phosphate buffered saline (PBS), glucose, glycerin, ethanol, etc., or a combination thereof. Other suitable carriers are well known to those of skill in the art. In addition, if desired, the composition can include trace amounts of auxiliaries such as wetting agents or emulsifiers, pH buffers.

The pharmaceutical composition according to the invention may be administered with the anti-neoplastic composition.

As used herein, the term "treatment" refers to both therapeutic and prophylactic or recurrence-preventing treatment. Those in need of treatment include those who already have a disability as well as those whose disability has been prevented.

The terms "cancer" and "cancerous" refer to or describe physiological diseases in mammals, typically characterized by disordered cell proliferation. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas and leukemias. More detailed examples of such cancers include lung cancer, thyroid cancer, breast cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, cervical cancer, pancreatic cancer, leukemia, lymphoma, myeloid leukemia, ovarian cancer. , Uterine cancer, sarcoma, biliary tract cancer, and endometrial cancer.

According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen that comprises the administration of at least one additional anti-cancer agent.

According to some embodiments, the anti-cancer agent comprises a metabolic antagonist, a mitotic inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, asparaginase, an alkylating agent, an antitumor antibiotic, and combinations thereof. Selected from the group. The individual possibilities represent separate embodiments of the invention.

According to some embodiments, the antimetabolite is selected from the group consisting of cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. According to some embodiments, the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine and vinorelbine. According to some embodiments, the topoisomerase inhibitor is selected from the group consisting of topotecan and irinotecan. According to some embodiments, the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambutyl, cyclophosphamide, cisplatin, carboplatin, ifosfamide, chlormethine, melphalan, thiotepa, dacarbazine, and procarbazine. NS. According to some embodiments, the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin. According to some embodiments, topoisomerase II is selected from the group consisting of etoposide and teniposide. The individual possibilities represent separate embodiments of the invention.

According to some specific embodiments, additional anti-cancer agents are selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof. The individual possibilities represent separate embodiments of the invention.

Monoclonal antibodies according to the invention may be used as part of a combination therapy with at least one anti-cancer agent. According to some embodiments, additional anti-cancer agents are immunomodulators, activated lymphocyte cells, kinase inhibitors or chemotherapeutic agents.

According to some embodiments, the anti-cancer agent is an immunomodulator, whether an agonist or an antagonist, such as an antibody against an immune checkpoint molecule.

Checkpoint-blocking immunotherapy has proven to be a new and exciting place for cancer treatment. The immune checkpoint pathway consists of a variety of co-stimulatory and co-suppressive molecules that work together to maintain self-tolerance under physiological conditions and protect tissues from damage by the immune system. Tumors utilize specific checkpoint pathways to evade the immune system. Therefore, inhibition of such pathways has become clear as a promising strategy for anti-cancer treatment.

The anti-cytotoxic lymphocyte 4 (CTLA-4) antibody ipilimumab (approved in 2011) was the first immunotherapeutic agent to be beneficial in the treatment of cancer patients. Antibodies interfere with inhibitory signals during antigen presentation to T cells. The anti-programmed cell death 1 (PD-1) antibody pembrolizumab (approved in 2014) blocks the negative immunoregulatory signal of the PD-1 receptor expressed by T cells. An additional anti-PD-1 agent was submitted in 2014 for regulatory approval for the treatment of non-small cell lung cancer (NSCLC). Active research is currently underway to explore many other immune checkpoints, including: CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activation gene 3 (LAG3). ), CD137, OX40 (also called CD134), and killer cell immune globulin-like receptors (KIR).

According to some embodiments, the immunomodulator is selected from the group consisting of: antibodies that inhibit CTLA-4, anti-human programmed cytotoxic protein 1 (PD-1), PD-L1 and PD-L2. Antibodies, active cytotoxic lymphocytes, lymphocyte activators, antibodies against CEACAM, antibodies against TIGIT, and RAF / MEK pathway inhibitors. The individual possibilities represent separate embodiments of the invention. According to some specific embodiments, additional immunomodulators are mAbs for PD-1, mAbs for PD-L1, mAbs for PD-L2, mAbs for CEACAM1, mABs for CTLA-4, mAbs for TIGIT, It is selected from interleukin 2 (IL-2) or lymphokine-activated killer (LAK) cells.

According to other embodiments, the additional anti-cancer agent is a chemotherapeutic agent. The chemotherapeutic agent could be administered with or separately from the antibody according to the invention, but may include, but is not limited to, any agent known in the art that exhibits anticancer activity, such as: May include: mitoxanthrone, topotecanase inhibitor, pyramidal inhibitor binca: binblastin, bincristine, binorerbin (taxol), paclitaxel, dosetacel; alkylating agents: mechloretamine, chlorambutyl, cyclophosphamide, melphalan, ifosphamide Metotrexate; 6-mercaptopurine; 5-fluorouracil, citarabin, gemcitabine; podophylrotoxins: etopocid, irinotecan, topotecan, daunorubicin; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosourea: carmustine (BCNU), lomustine (BCNU) , Epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxyphene, leuprolide, flutamide, and megestrol acetate.

According to some embodiments, the chemotherapeutic agent is an alkylating agent, a metabolic antagonist, a folic acid analog, a pyrimidine analog, a purine analog, and related inhibitors, a binca alkaloid, an epipodophylrotoxin, an antibiotic, L-Asparaginase, Topoisomerase Inhibitor, Interferon, Platinum Coordinator, Anthracendion Substitute Urea, Methylhydrazine Derivatives, Corticosuppressants, Corticosteroids, Progestin, Estrogen, Anti-Estrogen, Androgen, Anti-Androgen, and Gland Stimulating Hormone Release Selected from hormonal analogs. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel and docetaxel. One or more chemotherapeutic agents can be used.

In some embodiments, the pharmaceutical compositions of the present invention are used for the treatment of cancer or for enhancing the immune response.

The term "enhancing the immune response" refers to increasing the reactivity of the immune system and inducing or prolonging its memory. The pharmaceutical compositions of the present invention may be used to stimulate the immune system upon vaccination. Thus, in one embodiment, the pharmaceutical composition can be used to improve vaccination.

In certain embodiments, the cancers are lung cancer, thyroid cancer, breast cancer, colon cancer, melanoma, prostate cancer, liver cancer, bladder cancer, renal cancer, cervical cancer, pancreatic cancer, leukemia, lymphoma, myeloid leukemia, ovarian cancer. , Uterine cancer, sarcoma, biliary tract cancer, and endometrial cancer. The individual possibilities represent separate embodiments of the invention.

According to some embodiments, a pharmaceutical composition comprising at least one antibody or fragment thereof according to the invention, and a pharmaceutical composition comprising an additional immunomodulator or kinase inhibitor, treats cancer by separate administration. Used for.

According to yet another aspect, the invention provides a method of treating a subject's cancer in need, the method of which is a therapeutically effective amount of a monoclonal antibody or antibody fragment according to the invention to said subject. Includes the step of administering.

As used herein, the term "effective amount" refers to a sufficient amount of antibody fragment that has the intended therapeutic effect when administered to a subject. The effective amount required to achieve the final outcome of treatment is a number of factors, including, for example, the particular type of tumor and the severity of the patient's disease, and whether the combination is further co-administered with radiation. Can depend on. Effective amounts (dose) of the active agent, in the context of the present invention, include, but are not limited to, inhibition of tumor growth, reduction of the rate of tumor growth, prevention of growth of tumors and metastases and enhancement of survival of the subject. The amount should be sufficient to influence the beneficial therapeutic response over time.

The toxicity and therapeutic effects of the compositions described herein are standard in cell cultures or laboratory animals, for example by determining the maximum tolerability to IC50 (concentrations that provide 50% inhibition) and the subject compound. It can be determined by the pharmaceutical procedure of. The data obtained from these cell culture assays and animal studies can be used in dosage range formulations for use in humans. Dosage may depend, among other factors, on the dosage form used, the dosing regimen chosen, the composition of the drug used for the treatment and the route of administration used. The exact formulation, route of administration and dosage can be selected by the individual physician, taking into account the patient's illness. Depending on the severity and responsiveness of the disease to be treated, the dosing is also a single administration of the sustained release composition, the treatment process lasts days to weeks, or cure is achieved or the disease It can continue until the reduction of the condition is achieved. The amount of composition administered will, of course, depend on the subject being treated, the severity of the disease, the method of administration, the prescribing physician's judgment, and all other related factors.

The term "administering" or "administration of" a substance, compound or drug to a subject is performed using one of a variety of methods known to those of skill in the art. be able to. For example, a compound or drug can be administered intestine or parenterally. Intestinal refers to administration orally, sublingually or via the gastrointestinal tract, including the rectum. Parenteral administration is intravenous, intradermal, intramuscular, intraperitoneal, subcutaneous, eye, sublingual, intranasal, inhalation, intraspinal, intracerebral, and transdermal (by absorption, eg, skin duct). Includes administration by). The compound or agent is also properly introduced by a refillable or biodegradable polymer device or other device, such as a patch, pump, ie formulation, which provides sustained release, sustained release or controlled release of the compound or agent. can do. Administration can also be performed over a long period of time, eg, once, multiple times, and / or one or more times. In some embodiments, administration comprises both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who orders a patient to self-administer a drug or has another person administer a drug and / or provides a prescription for a drug to a patient. , Administer the drug.

Antibodies are generally administered in the range of about 0.1 mg / kg to about 20 mg / kg, generally 0.5 mg / kg to about 10 mg / kg, and frequently from about 1 mg / kg to about 5 mg / kg of the patient's weight. NS. In this regard, it is preferred to use antibodies having a circulating half-life of at least 12 hours, preferably at least 4 days, more preferably up to 21 days. Chimeric antibodies are expected to have a circulating half-life of up to 14-21 days. In some cases, it may be advantageous to administer large doses over the treatment period, followed by periodic (eg, weekly) maintenance doses. Antibodies can also be delivered by slow broadcast delivery systems, pumps, and other known delivery systems for continuous infusion.

The term "about" means that an acceptable error range for a particular value, eg, up to 5% or 10%, should be assumed.

<Angiogenesis>
According to one aspect, the invention provides a pharmaceutical composition according to the invention for use in the treatment of angiogenesis-related diseases or disorders.

Angiogenesis is an important cellular event in which vascular endothelial cells proliferate, prune, and reorganize to form new blood vessels from the pre-existing vascular network. There is compelling evidence that the development of vascular supply is essential for normal and pathological proliferative processes and inflammation. Vascular compartments are required not only for organ development and differentiation during embryogenesis, but also for adult wound healing, tissue remodeling and reproductive function.

Angiogenesis is also involved in the pathogenesis of various disorders, including, but not limited to, tumors, proliferative retinopathy, age-related macular degeneration, rheumatoid arthritis, and psoriasis.
Angiogenesis is essential for the growth of most primary tumors and their subsequent metastases.
Tumors can absorb sufficient nutrients and oxygen by simple diffusion up to a size of 1-2 mm, at which point their further growth requires meticulous vascular supply.
This process is thought to involve the recruitment of adjacent host mature vasculature to initiate the germination of new capillaries that grow towards the tumor mass and subsequently infiltrate it.
In addition, tumor angiogenesis involves the recruitment of circulating endothelial progenitor cells from the bone marrow to promote neoangiogenesis.

<Diagnosis>
The present invention further discloses methods for diagnosing and predicting cancer. According to one aspect, the invention provides a method of diagnosing and / or prognosis of a subject's cancer or infection, the method using at least one antibody as described herein. The step of determining the expression level of PVR in the biological sample of the subject is included.

The term "biological sample" includes various sample types obtained from an organism that can be used in diagnostic or monitoring assays. The term includes samples of blood and other liquids of biological origin, solid tissue samples such as biopsy specimens, or tissue cultures or cells derived from them and their progeny. In addition, the term may include circulating tumors or other cells. The term specifically embraces clinical samples and further includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, sheep water, aqueous humor and vitreous for eye samples. Includes biological fluids as well as tissue samples. The term also includes samples that have been manipulated in any post-procurement manner, such as treatment with reagents, solubilization, or enrichment of certain components.

Determining the expression level of PVR can be performed with labeled anti-PVR antibodies as described herein. The determination of expression can be performed, for example, by ELISA.

The methods of the invention can further include comparing the level of expression with the control level.

The following examples are presented to better illustrate some embodiments of the invention. They should not be construed as limiting the scope of the invention.

<Experimental procedure>
The following examples are referred to herein, which, together with the above description, illustrate the invention in a non-limiting manner.

In general, the terms used herein and the testing methods used in the present invention include molecular, biochemical, microbiology and recombinant DNA techniques. Such techniques are well known in the art. Other general references pointing to well-known procedures are provided throughout this document for the convenience of the reader.

Example 1. High PVR expression correlates with poor prognosis.
PVR and Nectin-2 are ligands for the inhibitory receptor TIGIT (Fig. 2). The results exemplify that high PVR expression levels correlated with poor prognosis for lung, breast and liposarcoma cancers (FIGS. 1A-1C, respectively). GEO expression of PVR is described in bioprofiling. Data that correlate with and relate to survival using de are IDs: GSE32101, GSE25055, GSE30929. Moreover, using the same analysis, Nectin-2 expression was mostly a positive marker for survival.

Example 2. Generation and Purification of Anti-PVR mAbs To produce anti-PVR antibodies, a recombinant protein, hPVR-Fc, is produced, which combines an extracellular portion of human PVR as an immunogen with the human Fc region of an immunoglobulin G carrier. Purified.

BALB / c mice were injected with 50 μg of immunogen in a complete Freund's adjuvant and after 2 weeks in an incomplete Freund's adjuvant. After 2 weeks, sera were screened for antibody titer. The best responders (serum monitored by ELISA assay for anti-hPVR-Fc antibody titers) had elevated immunogens in PBS. After 3 days, spleen cells were collected, lysed from erythrocytes and fused with SP2 / 0 cells. Cells were seeded in 20% RPMI 1640 medium containing hypoxanthine, aminopterin, and thymidine for hybridoma selection and screened for mAbs using ELISA. A stable hybridoma cell line was generated by fusing SP2 / 0 bone marrow cells with immunized mouse spleen cells.

Positive outcomes (cell lines that secrete antibodies that recognize hPVR-Fc) were further selected to develop products with some of the following distinguishing features: a) High yields to reduce the cost of antibody production. B) Lack of cross-reactivity between mouse and human PVRs and some other ligands for immune cell receptors; c) Natural, mature human PVR molecules expressed on the surface of living cells Strong binding ability to (antibodies selected from a comprehensive anti-hPVR monoclonal antibody pool with proven ability to recognize hPVR by different techniques such as flow cytometry, Western blot, ELISA). In fact, human and mouse PVRs have a high level of homology and it is not easy to produce mouse monoclonal antibodies that recognize human homologues. More importantly, human PVR is extensively glycosylated in the extracellular space of. For these reasons, it is not easy to generate antibodies that recognize intrinsic proteins using common antigens (such as those derived from Escherichia coli).

The present inventor uses the hPVR-Fc immunogen, a molecule produced in mammalian human embryonic kidney 293 (HEK 293T) cells and purified by immunoaffinity chromatography under native conditions. The natural protein, human PVR, was closely mimicked. In conclusion, from the pool of anti-hPVR mAbs generated, we identified typical ones that recognize the natural human PVR morphology on living cells, a derivative that can affect the human immune cell response during treatment. Is a prerequisite for developing.

Four anti-PVR antibodies were generated. Of these, three antibodies, namely antibody 5B9 (also called hPVR.09), antibody 7D4 (also called hPVR.01), and antibody 4E5 (also called hPVR.07) blocked anti-PVR mAbs. All of these antibodies block TIGIT-Ig binding (as exemplified by BD in FIG. 3, respectively). A fourth antibody that did not block TIGIT-Ig binding was generated and called 2G3 (also called hPVR.17) (A in FIG. 3).

Biosensor Biacore ™ T100 (GE Healthcare) from surface plasmon resonance (SPR) was used to determine Koff, kon and KD between the antibody and hPVR (Table 1).

Chimeric monoclonal antibodies comprising the human heavy chain constant IgG1 region specified in SEQ ID NO: 86 (corresponding to GenBank: AAA02914.1), using methods known in the art, the above three antibodies. Produced from.

Example 3. Blocking the PVR-TIGIT interaction with anti-PVR mAbs enhanced target cells for NK cell killing in human cell lines, labeled with [35S] -methionine 12 hours prior to assay. The indicated antibody was added to a final concentration of 5 μg / ml and incubated on ice for 30 minutes on a labeled target (5000 cells / well). The assay was performed in RPMI medium on a 96U plate at 37 ° C. for 5 hours. Labeled targets were incubated with effector NK cells in a 10: 1 E: T ratio. After incubation, the plates were centrifuged (1600 rpm, 5 minutes, 4 ° C.), the supernatant (50 μl) was collected and transferred to an opaque Opti plate (Packard). 150 μl of scintillation liquid (PerkinElmer) was added and analyzed by microbeta, β-counter (PerkinElmer). Maximum labeling was determined by adding 100 μl of 0.1 N NaOH to a uniform amount of no target (5000 / well). Spontaneous release was determined in wells containing only target cells. The final specific number of deaths was calculated as follows: ((radioactive reading-spontaneous release) / (maximum labeling-spontaneous release)) * 100 = specific number of deaths. As shown in FIG. 4, culturing NK cells in anti-PVR mAb 7D4 (also called hPVR.01) enhanced NK cell killing (2-fold) in the human mammary adenocarcinoma cell line MDA-MB-231. ..

Example 4. Blocking the PVR-TIGIT interaction with anti-PVR mAbs enhanced NK cell killing in human cancer cell lines Target cells were labeled with [35S] -methionine 12 hours prior to assay. The indicated antibody was added to a final concentration of 5 μg / ml and incubated on ice for 30 minutes on a labeled target (5000 cells / well). Cells were incubated with effector NK cells in a 10: 1 E: T ratio. The assay was performed in RPMI medium on a 96U plate at 37 ° C. for 5 hours. After incubation, the plates were centrifuged (1600 rpm, 5 minutes, 4 ° C.), the supernatant (50 μl) was collected and transferred to an opaque Opti plate (Packard). 150 μl of scintillation liquid (PerkinElmer) was added and analyzed by microbeta, β-counter (PerkinElmer). Maximum labeling was determined by adding 100 μl of 0.1 N NaOH to a uniform amount of no target (5000 / well). Spontaneous release was determined in wells containing only target cells. The final specific number of deaths was calculated as follows: ((radioactive reading-spontaneous release) / (maximum labeling-spontaneous release)) * 100 = specific number of deaths.

As shown in FIG. 5, blocking the PVR-TIGIT interaction with anti-PVR mAb 4E5 (also called hPVR.07) enhanced NK cell killing in the human cancer cell line HepG2 (hepatocellular carcinoma). Therefore, it is clear that blocking PVR leads to enhanced killing of target cells. Death is further enhanced when a chimeric version of mAb is used for the human IgG counterpart of 4E5. Death is equivalent to a positive control (Erbitux (copyright)).

Example 5. Human tumor cell lines express PVR and Nectin-2. To test the expression of PVR and Nectin-2 on tumor cells, the expression levels of these proteins on different tumor cell lines were adjusted to anti-PVR-4E5 Ab. And anti-Nectin-2 Ab (clone TX-31), both tested at 2 μg / ml by FACS analysis. As shown in FIGS. 6A-6O, various human tumor cell lines express PVR and Nectin-2. Specifically, melanoma cells (Fig. 6A-6E), breast cancer cells (Fig. 6F-6H), colon cells (Fig. 6I), kidney cells (Fig. 6J), lung cancer cells (Fig. 6K), prostate cancer cells (Fig. 6L). ), Brain tumor cells (Fig. 6M), and hepatocellular carcinoma cells (Fig. 6N-6O) have all been shown to express PVR and Nectin-2.

Example 6. PVR is the major TIGIT ligand The AC in FIG. 7 demonstrates that PVR is the major TIGIT ligand. Specifically, HepG2 cells (human hepatocellular carcinoma cells) were shown to express both PVR and Nectin-2 (A in FIG. 7). hPVR. Culturing HepG2 cells in purified anti-PVR mAb 4E5, also called 07 (0.15 μg / well), resulted in TIGIT-Ig binding (2 μg / well) despite the fact that these cells also express Nectin-2. ml) was almost completely blocked (B in FIG. 7). It is clear that there was no direct recognition of Nectin-2 by anti-PVR mAbs, as shown in C of FIG. 7 and D of FIG.

Example 7. Binding of mAb clones to human and primate PVRs As shown in FIGS. 8A-C, all anti-PVR antibody clones tested bind to human PVR (hPVR) using FACS analysis. Briefly, cells were trypsinized for staining with 2 ^* 10 ⁵ cells per well was transferred. The indicated antibody was added on ice for 30 minutes. All antibodies were used at a final concentration of 2 μg / ml. Detection of bound anti-PVR Ab was performed using anti-mouse IgG-647. Mouse IgG1 kappa was used as a negative control.

FIG. 8A illustrates that endogenous hPVR was detected by FACS staining of the surface of HepG2 hepatocellular carcinoma cells. In FIG. 8B, mouse cell line B16 was used. Mouse PVR sequences, unlike humans, are not recognized by anti-human PVR antibodies (left panel) as seen in the lack of signaling. When the full-length hPVR protein (NP_006496.64 amino acids 1-418) was overexpressed in these cells, all three clones resulted in the same signal (right panel), which is the result of this staining. It further supports the claim that it is the result of the specific binding of these Abs to human PVR proteins.

The PVR amino acid sequence is conserved across several species. The amino acid preservation of human PVR was compared to the amino acid preservation of African green monkeys in silico and found to have 93% similarity to human PVR. FACS staining of Vero cells in African green monkeys demonstrated that PVRs in African green monkeys were efficiently recognized by all three human mAbs (Fig. 8C). It was further found that these human anti-PVR antibodies do not recognize PVR from canines such as hamsters or mice and rodents. FIG. 9 shows FACS analysis for PVR in canines expressing MDCK cells. As can be seen, none of the anti-human Abs resulted in a positive signal, while PVR expression itself is suggested by a strong TIGIT-Ig signal.

Example 8. Nectin-2 stained cells overexpressing hNectin-2 using TIGIT-Fc and DNAM-1-Fc, which are preferentially bound by DNAM-1, at the concentrations shown in FIG. -8866 cell lines (FIGS. 10A and 10B), or B16-hPVR cell lines (FIGS. 10C and 10D). Detection of bound fusion proteins was performed using anti-human IgG APC and analyzed by FACS.

Regarding PVR binding, the signal by TIGITT-Fc is 2 from the signal of DNAM-1-Fc, as in the previous report (Yu. X et al., 2009, Nat Immunology, 10 (1): 48-57). -4 times higher. At the same time, the binding of hNectin-2 to DNAM-1-Fc is 2-10 times stronger than its binding to TIGIT-Fc, which is inconsistent with one report (Yu.X et al 2009). , Another test (Zhu Y et al., 2016, J Exp Med., 8; 213 (2): 167-76). Taken together, the results exemplify that DNAM-1 is the preferred receptor for Nectin-2 binding. Thus, blocking PVR prevents TIGIT inhibitory signaling while allowing co-stimulation signaling by DNAM-1. DNAM-1 mediates cell adhesion to other cells that carry its ligand.

Example 9. Anti-PVR antibody enhances T cell proliferation To test the effect of anti-PVR mAb on T cell proliferation, human peripheral blood mononuclear cells (PBMC) were stained with carboxyfluorescein succinimidyl ester (CFSE), 4 μg. Incubated in target cells in the presence of antibody at a concentration of / ml. CFSE dilution was measured on CD45 positive cells 5-9 days after culturing. As shown in FIG. 11, anti-PVR 5B9 activity exceeds the antibody activity of PD-1 and CTLA4 as a single agent. Also, the chimeric anti-PVR 4E5hIgG1 clone having the human IgG1 constant region was superior to its mouse counterpart. Next, the combined effect of anti-PVR mAbs and other antibodies found to enhance T cell proliferation was examined. Human PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and incubated in target cells at a concentration of 4 μg / ml in the presence of antibody. CFSE dilution was measured on CD45 positive cells 5-9 days after culturing. The results show that the proliferative activity of anti-PVR 5B9 exceeds the activity of the combination of PD-1 and CTLA4 when used in combination with either PD-1 or CTLA-4 (FIG. 12). Also, the activity of the combination of anti-PVR 4E5 and CTLA-4 is equal to that of the combination of PD-1 and CTLA4. Next, the specific induction of CD8 was examined. As shown in FIG. 13, the T cell proliferative activity of the anti-PVR antibody CD8 exceeds that of PD1. Also, the evoked activity of the anti-PVR 5B9 antibody exceeds the evoked activity of CTLA1. The ratio of CD8 / CD4 proliferation was evaluated for different antibodies (FIG. 14). Anti-PVR 5B9 had the highest CD8 / CD4 ratio. The variable heavy chain and variable light chain DNA sequences were used to construct chimeric antibodies containing a human IgG1 isotype constant domain and a constant light (CL) human IgG kappa domain. T cell proliferation induced by antibodies to the mouse and human chimeric counterparts was measured by CFSE assay. The numbers shown in Table 2 represent the relative levels of proliferation compared to controls.

Next, the effect of PVR antibody on NK degranulation was examined. During degranulation, cytolytic granules in NK cells are released, and lysosome-related membrane proteins-1 (LAMP-1, CD107a) present on the surface of the cytolytic granules are transported to the cell surface to form an antibody. Joins are available. This marker allows the identification of activated NK cells. NK cells were incubated with anti-PVR antibodies (antibodies of mice and their chimeric human counterparts) in 5 different target cells. Degranulation was evaluated using anti-CD107a antibody.

As shown in Table 3 and FIG. 15, all antibodies exhibited NK degranulation activity, where the chimeric antibody had significantly higher activity compared to their corresponding mouse antibodies.

Example 10. Anti-PVR antibodies reduce the viability of tumor cells in the absence of immune cells.
Cell viability of A549, U373, HCT116, and Mel-624 was examined using the MTT cell viability assay in the presence of 50 micrograms / ml different anti-PVR mAbs for 24 hours. As shown in FIGS. 16A-16D and Table 4, PVRs blocked by 5B9 mAbs significantly reduced survival by 20-40% compared to mIgG.

Example 11. In vivo Effect of Anti-PVR Antibodies on Human Tumors in Humanized Mouse Models-Short-term antitumor effects of humanized antibodies will be tested in vivo. To assess the effect of the antibodies described herein in inhibiting human cancer, the antibodies are tested in a model that combines both tumors of human origin and lymphocytes. NOD scid gamma (NSG) mice are transplanted with hPBMC to restore immune eligibility and challenge with human cancer cells. At a predetermined time point / tumor size, mice are treated with anti-human PVR antibodies according to the invention by administration of multiple doses at different time points after tumor attack. The same experiment is performed with a chimeric anti-PVR antibody. Tumor growth curves and body weight are measured at 3x / week and extensive phenotypic analysis of TIL and immune populations in different organs is performed after sacrifice of mice.

A model similar to the tumor strain in PBMC huNSG mice was developed by Gupta P. et al. , Oncoimmunology. 2015 Mar 6; 4 (2): Perform according to e981449.

Example 12. In vivo Effect of Anti-PVR Antibodies on Human Tumors in Humanized Mouse Models-To assess the effects of anti-PVR antibodies on inhibition of long-term humanized human cancer, antibodies were used for tumors and lymphocytes of human origin. Test in a model that combines both. Newborn NOD scid gamma (NSG) puppies are exposed to radiation and transplanted with CD34 + HSC to restore immune eligibility. 1-2 weeks after the determination of immune cell rearrangement, mice were loaded with human cancer cells and mice were subjected to multiple times at predetermined time points / tumor sizes at different time points with the anti-human PVR antibody according to the invention. Treat by administration of dose. Perform the same experiments as humanized anti-PVR antibodies. Tumor growth curve and body weight are measured at 3x / week. When mice are sacrificed, extensive phenotypic analysis of TIL and immune populations in different organs is performed.

A similar model used to test the tumor suppressive activity of the antibodies of the invention is The Jackson Laboratory (http://immune-checkpoint.com/pp-content/uploads/sites/24/2015/01/1/Day). It was established by -1-15.45-Rick-Huntress.pdf).

Example 13. Inducing IFNγ secretion To test the effect of anti-PVR mAbs on cytokine secretion, antibodies from human peripheral blood mononuclear cells (PBMC) from two healthy donors at concentrations of 1 μg / ml and 0.1 μg / ml. Incubated in target cells in the presence of (mIgG, 5B9mIgG, anti-CTL-4 antibody called ipilimumab). The level of IFNγ was measured 6 days after culturing. Significant induction of IFNγ by anti-PVR 5B9 antibody was observed (P = 7.34548E- ^{11 for 1 μg / ml and 2.73179E-08} for 0.1 μg / ml).

IFNγ secretion is a major component of antitumor immunity, and induction of IFNγ secretion by anti-PVR mAbs exhibits a potential additional antitumor effect of these compounds in cancer treatment.

The above description of a specific embodiment fully clarifies the general nature of the invention in which others can be made by applying current knowledge and deviates from similar concepts without unnecessary experimentation. Without doing so, such specific embodiments are readily modified and / or applied for a variety of uses, and therefore such applications and modifications are meant to be equivalents of the disclosed embodiments. And should be understood within scope and is intended as such. It should be understood that the terminology or terminology used herein is for explanatory purposes only and is not limiting.

Claims (14)

An isolated monoclonal antibody that binds to a human poliovirus receptor (PVR), or an antibody fragment thereof containing at least an antigen-binding moiety, comprising a CDR set selected from the group consisting of a CDR set of 6 CDRs. wherein, HC CDRl is, GYTFSNYWIE (SEQ ID NO: 36 ) and SNYWIE (SEQ ID NO: 84) is selected from, HC CDR2 is EIFPGSGRINFNEKFKG (SEQ ID NO: 38) and is, HC CDR3 is TKIYGNSFDY (SEQ ID NO: 40) , LC CDR1 is selected from KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85) , and LC CDR2 is WASSRHN (SEQ ID NO: 46), WASSRHA (SEQ ID NO: 85). : 56), from WASSRHR (SEQ ID NO: 57), WASSRHD (SEQ ID NO: 58), WASSRHE (SEQ ID NO: 59), WASSRHP (SEQ ID NO: 60), and WASSRHT (SEQ ID NO: 61). is selected from the group consisting, and, LC CDR3 is QQYSRYPLT: a (SEQ ID NO 48), wherein the isolated monoclonal antibodies, PVR T cell immunoreceptor with Ig and ITIM domains for (TIGIT) An isolated monoclonal antibody or antibody fragment thereof that can prevent the binding of the antibody. The HC CDR1 sequence is selected from the group consisting of GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84), the HC CDR2 sequence consists of EIFFGSGRINFNEKFKG (SEQ ID NO: 38), and the HC CDR3 is sequence: TKY. The LC CDR1 sequence consists of (SEQ ID NO: 40) and the LC CDR1 sequence is selected from the group consisting of KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85), and the LC CDR2 sequence: WASSRHE (SEQ ID NO: 85). : 59), and LC CDR3 is the isolated monoclonal antibody according to claim 1, or an antibody fragment thereof, which comprises the sequence: QQYSRYPLT (SEQ ID NO: 48). One of the isolated monoclonal antibody of claim 1 or 2, or, an isolated polynucleotide encoding the antibody fragments. Plasmid containing an isolated polynucleotide of claim 3. Hybridoma cells comprising the isolated polynucleotide according to any one of claims 3 or 4. A hybridoma cell capable of producing the monoclonal antibody according to any one of claims 1 or 2. The monoclonal antibody according to claim 1 or 2 , or an antibody fragment thereof, which is attached to a cytotoxic part, a radioactive part, or an identifiable part. A pharmaceutical composition comprising, as an active ingredient, the isolated monoclonal antibody according to claim 1 or 2 , or an antibody fragment thereof, and a pharmaceutically acceptable excipient, diluent, salt, or carrier. thing. The pharmaceutical composition according to claim 8, which is used in regulating the immune system by inhibiting the binding of PVR to TIGIT. The pharmaceutical composition according to claim 8, for use in the treatment of cancer. The pharmaceutical composition according to claim 10, wherein the use further comprises administering to the subject an additional immunomodulatory agent, activated lymphocyte cells, a kinase inhibitor, a chemotherapeutic agent, or another anticancer agent. .. Additional immunomodulators include PD-1, CTLA-4, PDL-1, CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activation gene 3 (LAG3), CD137, OX40 (CD134). The pharmaceutical composition according to claim 11, which is an antibody against an immune checkpoint molecule selected from the group consisting of (also referred to as), killer cell immunoglobulin-like receptor (KIR), TIGIT, and any combination thereof. The pharmaceutical composition according to claim 10, wherein the cancer is a solid cancer. The pharmaceutical composition according to claim 10, wherein the cancer is a cancer of blood.

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